Novel receptor

ABSTRACT

The present invention relates to the novel GABA B  receptor subtypes GABA B -R1c and GABA B -R2 as well as to a novel, functional GABA B  receptor which comprises a heterodimer of GABA B -R1 and GABA B -R2 receptor subunits. The present invention also relates to variants of the receptors, nucleotide sequences encoding the receptors and variants thereof and novel vectors, stable cell lines, antibodies, screening methods, methods of treatment and methods of receptor production.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This US patent application claims priority to GB9819420.2 filedon Sep. 7, 1998 in the United Kingdom and to U.S. provisionalapplication No. 60/103,670 filed on Oct. 9, 1998 in the United StatesPatent Office.

FIELD OF THE INVENTION

[0002] The present invention relates to the novel GABA_(B) receptorsubtypes GABA_(B)-R1c and GABA_(B)-R2 as well as to a novel, functionalGABA_(B) receptor which comprises a heterodimer of GABA_(B)-R1 andGABA_(B)-R2 receptor subunits. The present invention also relates tovariants of the receptors, nucleotide sequences encoding the receptorsand variants thereof and novel vectors, stable cell lines, antibodies,screening methods, methods of treatment and methods of receptorproduction.

BACKGROUND OF THE INVENTION

[0003] GABA (γ-amino-butyric acid) is the main inhibitoryneurotransmitter in the central nervous system (CNS) activating twodistinct families of receptors; the ionotropic GABA_(A) and GABA_(C)receptors for fast synaptic transmissions, and the metabotropic GABA_(B)receptors governing a slower synaptic transmission. GABA_(B) receptorsare members of the superfamily of 7-transmembrane G protein-coupledreceptors. Activation results in signal transduction through a varietyof pathways mediated principally via members of the G_(i)/G_(o) familyof pertussis toxin-sensitive G proteins. GABA_(B) receptors have beenshown to inhibit N, P/Q and T-type Ca²⁺ channels in a pertussistoxin-sensitive manner (Kobrinsky et al., 1993; Menon-Johansson et al.,1993; Harayama et al., 1998) and indeed there is also some evidence fordirect interactions between GABA_(B) receptors and Ca²⁺ channels sinceCa²⁺ channel ligands can modify the binding of GABA_(B) agonists (Ohmoriet al., 1990). GABA_(B) receptor-mediated Ca²⁺ channel inhibition is theprinciple mechanism for presynaptic inhibition of neurotransmitterrelease. Post-synaptically the major effect of GABA_(B) receptoractivation is to open potassium channels, to generate post-synapticinhibitory potentials.

[0004] Autoradiographic studies show that GABA_(B) receptors areabundant and heterogeneously distributed throughout the CNS, withparticularly high levels in the molecular layer of the cerebellum,interpeduncular nucleus, frontal cortex, olfactory nuclei and thalamicnuclei. GABA_(B) receptors are also widespread in the globus pallidus,temporal cortex, raphe magnus and spinal cord (Bowery et al., 1987).GABA_(B) receptors are an important therapeutic target in the CNS forconditions such as spasticity, epilepsy, Alzheimer's disease, pain,affective disorders and feeding. GABA_(B) receptors are also present inthe peripheral nervous system, both on sensory nerves and onparasympathetic nerves. Their ability to modulate these nerves givesthem potential as targets in disorders of the lung, GI tract and bladder(Kerr and Ong, 1995; 1996; Malcangio and Bowery, 1995).

[0005] Despite the widespread abundance of GABA_(B) receptors,considerable evidence from neurochemical, electrophysiological andbehavioural studies suggests that multiple subtypes of GABA_(B)receptors exist. This heterogeneity of GABA_(B) receptors may allow thedevelopment of selective ligands, able to target specific aspects ofGABA_(B) receptor function. This would lead to the development of drugswith improved selectivity profiles relative to current compounds (suchas baclofen) which are relatively non-selective and show a variety ofundesirable behavioural actions such as sedation and respiratorydepression. Multiple receptor subtypes are best classified by thediffering profiles of agonist and antagonist ligands.

[0006] To date screening for GABA_(B) ligands and subsequentstructure/activity determinations has relied on radioligand bindingassays to rat brain membranes. Further analysis of such ligands inanimal models has indicated differences in their behavioural profile.However, due to the absence of cloned GABA_(B) receptors the molecularbasis for such differences has not been defined, and therefore it hasnot been possible to optimise GABA_(B) ligands for therapeutic use.

[0007] GABA_(B) receptors were first described nearly 20 years ago (Hilland Bowery, 1981), but despite extensive efforts using conventionalexpression cloning strategies, for example in Xenopus oocytes, orcloning based on sequence homology, the molecular nature of the GABA_(B)receptor remained elusive. The development of a high affinity antagonistfor the receptor finally allowed Kaupmann et al., (1997) to expressionclone the receptor from a rat cerebral cortex cDNA using a radioligandbinding assay. Two splice variants of the receptor were identified,GABA_(B)-R1a encoding a 960 amino acid protein and GABA_(B)-R1b,encoding an 844 amino acid protein, differing only in the lengths oftheir N-termini. These two splice variants have distinct spatialdistributions within the brain, but both reside within neuronal ratherthan glial cells. Pharmacologically, the two splice variants aresimilar, showing binding affinities for a range of antagonists, butabout 10 fold lower than those of native receptors, as well as agonistdisplacement constants which are about 100-150 fold lower than those ofnative receptors. These observations have led to speculation that thecloned receptor was a low affinity receptor and an additional highaffinity, pharmacologically distinct GABA_(B) receptor subtype couldexist in the brain. Alternatively, it was argued that G-protein couplingwas inefficient or the receptor was desensitising in the recombinantsystems used.

[0008] A number of groups working in the area have, however, found thatthe cloned receptor fails to behave as a functional GABA_(B) receptoreither in mammalian cells or in Xenopus oocytes. The present inventiondescribes the cloning of a novel human GABA_(B) receptor subtype,GABA_(B)-R2, the identification of a novel splice variant GABA_(B)-R1c,and the surprising observation that GABA_(B)-R1 and GABA_(B)-R2 stronglyinteract via their C-termini to form heterodimers. Co-expression ofGABA_(B)-R1 and GABA_(B)-R2 allows trafficking of GABA_(B)-R1 to thecell surface and results in a high affinity functional GABA_(B) receptorin both mammalian cells and Xenopus oocytes.

[0009] These surpising findings provide a unique opportunity to defineGABA_(B) subtypes at the molecular level, which in turn will lead to theidentification of novel subtype-specific drugs.

SUMMARY OF THE INVENTION

[0010] According to one embodiment of the present invention there isprovided an isolated GABA_(B)-R2 receptor protein or a variant thereof.

[0011] According to another embodiment of the invention there isprovided an isolated GABA_(B)-R2 receptor protein having amino acidsequence provided in FIG. 1B, or a variant thereof.

[0012] According to a further embodiment of the invention there isprovided a nucleotide sequence encoding a GABA_(B)-R2 receptor or avariant thereof, or a nucleotide sequence which is complementarythereto.

[0013] According to a further embodiment of the invention there isprovided a nucleotide sequence encoding a GABA_(B)-R2 receptor, as shownin FIG. 1A, or a variant thereof, or a nucleotide sequence which iscomplementary thereto.

[0014] According to a further embodiment of the invention there isprovided an expression vector comprising a nucleotide sequence asreferred to above which is capable of expressing a GABA_(B)-R2 receptorprotein or a variant thereof.

[0015] According to a still further embodiment of the invention there isprovided a stable cell line comprising a vector as referred to above.

[0016] According to another embodiment of the invention there isprovided an antibody specific for a GABA_(B)-R2 receptor protein or avariant thereof.

[0017] According to another embodiment of the invention there isprovided an isolated GABA_(B)-R1c receptor protein or a variant thereof.

[0018] According to another embodiment of the invention there isprovided an isolated GABA_(B)-R1c receptor protein having amino acidsequence provided in FIG. 2, or a variant thereof.

[0019] According to another embodiment of the invention there isprovided a nucleotide sequence encoding a GABA_(B)-R1c receptor proteinor a variant thereof, or a nucleotide sequence which is complementarythereto.

[0020] According to another embodiment of the invention there isprovided an expression vector comprising a nucleotide sequence asreferred to above, which is capable of expressing a GABA_(B)-R1creceptor protein or a variant thereof.

[0021] According to another embodiment of the invention there isprovided a stable cell line comprising a vector as referred to above.

[0022] According to a further embodiment of the invention there isprovided an antibody specific for a GABA_(B)-R1c receptor protein or avariant thereof.

[0023] According to a further embodiment of the invention there isprovided a GABA_(B) receptor comprising an heterodimer between aGABA_(B)-R1 receptor protein or a variant thereof and a GABA_(B)-R2receptor protein or a variant thereof.

[0024] According to a further embodiment of the invention there isprovided an expression vector comprising a nucleotide sequence encodingfor a GABA_(B)-R1 receptor or a variant thereof and a nucleotidesequence encoding for a GABA_(B)-R2 receptor or variant thereof, saidvector being capable of expressing both GABA_(B)-R1 and GABA_(B)-R2receptor proteins or variants thereof.

[0025] According to a further embodiment of the invention there isprovided a stable cell line comprising a vector as referred to above.

[0026] According to a further embodiment of the invention there isprovided a stable cell line modified to express both GABA_(B)-R1 andGABA_(B)-R2 receptor proteins or variants thereof.

[0027] According to a further embodiment of the invention there isprovided a GABA_(B) receptor produced by a stable cell line as referredto above.

[0028] According to a further embodiment of the invention there isprovided an antibody specific for a GABA_(B) receptor as referred toabove.

[0029] According to a further embodiment of the invention there isprovided a method for identification of a compound which exhibitsGABA_(B) receptor modulating activity, comprising contacting a GABA_(B)receptor as referred to above with a test compound and detectingmodulating activity or inactivity.

[0030] According to a further embodiment of the invention there isprovided a compound which modulates GABA_(B) receptor activity,identifiable by a method as referred to above.

[0031] According to a further embodiment of the invention there isprovided a method of treatment or prophylaxis of a disorder which isresponsive to modulation of GABA_(B) receptor activity in a mammal,which comprises administering to said mammal an effective amount of acompound identifiable by the method referred to above.

BRIEF DESCRIPTION OF THE FIGURES

[0032]FIGS. 1A (2 pages) and 1B. Nucleotide and protein sequences ofHuman GABA_(B)-R2

[0033] Nucleotide sequence (a) and the translated protein sequence (b)for Human GABA_(B)-R2 are shown.

[0034]FIG. 2 (3 pages). Protein alignments between GABA_(B)-R1a,GABA_(B)-R1b, GABA_(B)-R1c splice variants and GABA_(B)-R2.

[0035] Amino-acid sequences of the human GABA_(B)-R1a, GABA_(B)-R1b andGABA_(B)-R2 receptors aligned for comparison. Signal sequences andpredicted cleavage point (

), together with the N-terminal splice points for GABA_(B)-R1a andGABA_(B)-R1b are shown. GABA_(B)-R1c sequence is exactly that ofGABA_(B)-R1a, except for the deletion of 63 amino acids (open box).Amino acids conserved between GABA_(B)-R1a and GABA_(B)-R1b are in boldtype and potential N-glycosylation sites (*) are shown. Lines beneaththe text show positions of the seven predicted TM domains and regionsencoding coiled coil structure are indicated by shading. The C-terminalregion of GABA_(B)-R1 used as the bait in the yeast two hybrid analysisis marked as ‘BAIT→’, and GABA_(B)-R2 C-terminal domains recovered fromthe library screen against GABA_(B)-R1 C-terminus are shown as ‘YTHHITS→’.

[0036]FIG. 3. Hydrophobicity profile of GABA_(B)-R2.

[0037] Hydrophobicity profiles of GABA_(B)-R2 sequence were determinedusing the Kyte-Doolittle algorithm, whereby positive values indicatehydrophobic regions. The predicted signal sequence and seventrans-membrane domains are shown.

[0038]FIGS. 4A and 4B. Tissue Distribution Studies for Human GABA_(B)-R1and GABA_(B)-R2.

[0039] A Human RNA Master Blot (Clontech), containing normalised polyA⁺mRNA from multiple tissues of adult and fetal origin, were probedsequentially with a pan specific probe for GABA_(B)-R1 (all splicevariants) followed by a GABA_(B)-R2 specific probe. Resultingautoradiographic analysis of the blots are shown, together with a grididentifying tissue type. Specificity controls include yeast RNA and E.coli DNA.

[0040]FIG. 5. Heterodimerisation and homodimerisation between theC-terminal domains of the GABA_(B)-R1 and GABA_(B)-R2 receptors in theyeast two hybrid system.

[0041] β-galactosidase activity was measured in yeast Y190 cellsexpressing the GABA_(B)-R1 or the GABA_(B)-R2 C-termini, either againstempty vector or against each other in all combinations, using ONPG. Ofeach pair of proteins expressed in the two hybrid system, the firstalways refers to the GAL4_(BD) fusion construct whilst the second refersto the GAL4_(AD) fusion construct. β-galactosidase activity isdetermined relative to cell numbers and is in arbitary units.

[0042]FIG. 6. Co-immunoprecipitation studies of the GABA_(B) heterodimerin HEK239 cells.

[0043] HEK293T cells were transfected with 1 μg each of eitherMyc-GABA_(B)-R1b or HA-GABA_(B)-R2 alone or in combination. Cells wereharvested 48 h after transfection, lysed and epitope tagged receptorsimmunoprecipitated using 12CA5 (HA) or 9E10 (Myc) antisera as describedin Methods. Immune complexes were then subjected to SDS-PAGE,transferred to nitrocellulose, and captured Myc-GABA_(B)-R1b andHA-GABA_(B)-R2 identified by immunoblotting with Myc and HA,respectively. Lanes 1 and 4, immunoprecipitates of cells transfectedwith Myc-GABA_(B)-R1b only; lanes 2 and 5, HA-GABA_(B)-R2 only; lanes 3and 6, immunoprecipitates of cells transfected with Myc-GABA_(B)-R1btogether with HA-GABA_(B)-R2. Lanes 1-3, lysates immunoprecipitated with9E10 (Myc) and blotted to 12CA5(HA); lanes 4-6, lysatesimmunoprecipitated with 12CA5(HA) and blotted with 9E10 (Myc)

[0044]FIG. 7. Cell surface localisation of GABA₆-R1 receptor isdependent upon coexpression with GABA_(B)-R2.

[0045] Flow cytometry was performed on HEK293T cells transfected with 1μg of either Myc-GABA_(B)-R1b or HA-GABA_(B)-R2 or both receptors, incombination. (A) Analysis using 9E10 (c-Myc) as primary antibody todetect Myc-GABA_(B)-R1b; intact cells. (B) Analysis using 9E10 (c-Myc)as primary antibody to detect Myc-GABA_(B)-R1b; permeabilised cells. (C)Analysis using 12CA5 (HA) as primary antibody to detect HA-GABA_(B)-R2;intact cells. Mock transfected cells, reflecting backgroundfluorescence, are shaded and the marker indicates fluorescence measuredover background levels. Myc-GABA_(B)-R1b data is shown as a grey linewhereas co-expression of Myc-GABA_(B)-R1b with HA-GABA_(B)-R2 is shownin black. 30,000 cells were analysed in each sample. Histograms shownare from a single experiment. Quoted statistics are from mean of threeseparate transfections and analysis.

[0046]FIG. 8. Coexpression of GABA_(B)-R1a and 1b splice variants withGABA_(B)-R2 receptors in HEK293T cells results in terminal glycosylationof both GABA_(B)-R1a and GABA_(B)-R1b. P2 membrane fractions werederived from HEK293T cells that were transfected with 1 μg of eitherGABA_(B)-R1a (lanes 1-3), GABA_(B)-R1b (lanes 4-6) or HA-GABA_(B)-R2(lanes 13-15), or with 1 μg each of HA-GABA_(B)-R2 in combination with 1μg of either GABA_(B)-R1a (lanes 7-9, 16-18) or GABA_(B)-R1b (lanes10-12, 19-21). Glycosylation status of transfected receptors wasassessed following treatment of P2 fractions (50 μg of membrane protein)with either vehicle (lanes 1, 4, 7, 10, 13, 16 and 19), endoglycosidaseF (lanes 2, 5, 8, 11, 14, 17 and 20) or endoglycosidase H (lanes 3, 6,9, 12, 15, 18 and 21). Samples were resolved by SDS-PAGE (10% (w/v)acrylamide), transferred to nitrocellulose, and immunoblotted. Upperpanel, antiserum 501 was used as primary reagent to allow identificationof both GABA_(B)-R1a and 1b. Lower panel, 12CA5 anti-HA antiserum wasemployed to identify HA-GABA_(B)-R2. *, denotes terminally glycosylatedforms of GABA_(B)-R1a and 1b.

[0047]FIGS. 9A and 9B. Coexpression of GABA_(B)-R1 and GABA_(B)-R2receptors in HEK293T cells leads to GABA-mediated stimulation of[³⁵S]GTPγS binding activity.

[0048] [³⁵S]GTPγS binding activity was measured on P2 particulatefractions derived from HEK293T cells transfected with 1 μg of G_(o1)αtogether with 1 μg of either GABA_(B)-R1a, GABA_(B)-R1b orHA-GABA_(B)-R2; or with 1 μg each of G_(o1)α and HA-GABA_(B)-R2 incombination with 1 μg of either GABA_(B)-R1a or GABA_(B)-R1b. (A)[³⁵S]GTPγS binding was measured in the absence (open bars) or presence(hatched bars) of GABA (10 mM) as described in Methods. (B) The abilityof varying concentrations of GABA to stimulate the binding of [³⁵S]GTPγSwas measured on P2 membrane fractions from HEK293T cells expressingeither G_(o1)α and HA-GABA_(B)-R2 alone (open circles) or in combinationwith either GABA_(B)-R1a (closed squares) or GABA_(B)-R1b (closedtriangles). The data shown are the means±S.D. of triplicate measurementsand are representative of three independent experiments.

[0049]FIG. 10. GABA-mediated stimulation of [³⁵S]GTPγS binding activityin HEK293T cells coexpressing GABA_(B)-R1 and GABA_(B)-R2 receptorsrequires cotransfection with additional G_(I) G protein, G_(o1)α.

[0050] [³⁵S]GTPγS binding activity was measured on P2 particulatefractions derived from HEK293T cells transfected with HA-GABA_(B)-R1b (1μg) together with HA-GABA_(B)-R2 (1 μg) and G_(o1)α (11g) (closedtriangles), or in combination with either HA-GABA_(B)-R2 (1 μg) (opencircles) or G_(o1)α (1 μg) (closed circles). The ability of varyingconcentrations of GABA to stimulate the binding of [³⁵S]GTPγS wasdetermined. Data shown are the mean±S.D. of triplicate measurements.

[0051]FIGS. 11A and 11B. Coexpression of GABA_(B)-R1 and GABA_(B)-R2receptors in HEK293T cells permits GABA-mediated inhibition offorskolin-stimulated adenylate cyclase activity

[0052] cAMP levels were measured in HEK293T cells transfected with 1 μgof G_(i1)α together with 1 μg of either GABA_(B)-R1a, GABA_(B)-R1b orHA-GABA_(B)-R2; or with 1 μg each of G_(i1)α and HA-GABA_(B)-R2 incombination with 1 μg of either GABA_(B)-R1a or GABA_(B)-R1b, asdescribed in Methods. (A) cAMP levels were determined in cells treatedwith forskolin (50 μM) in the absence (open bars) or presence (hatchedbars) of GABA (1 mM). (B) ability of varying concentrations of GABA toinhibit forskolinelevated adenylate cyclase activity in HEK293T cellsexpressing G_(i1)α and HA-GABA_(B)-R2 in combination with GABA_(B)-R1b.The data shown are the means±S.D. of triplicate measurements.

[0053]FIGS. 12A and 12B. Co-expression of GABA_(B)-R1 and GABA_(B)-F2receptors in Xenopus oocytes permits agonist-dependant activation of ionflux through CFTR and GIRK1/4.

[0054] Xenopus oocytes were injected with CRNA encoding GABA_(B)-R1 andGABA_(B)-R2 receptors (in equal amounts for CFTR, 1:2 ratio for GIRK)plus either CFTR (A) or the GIRK1/GIRK4 heteromer (B). A, Time courseplot for an oocyte expressing GABA_(B)-R1, GABA_(B)-R2 and CFTR.Application of 10 mM GABA, 100 mM SKF97541 or 1 mM Baclofen (arrows)activated a large inward CFTR current. Note the increase in CFTRresponse seen with repeated GABA application. B, Time course plot for anoocyte expressing GABA_(B)-R1, GABA_(B)-R2, GIRK1 and GIRK4. Switchingfrom ND96 (low potassium) to 90K (high potassium) solution led to aninward shift in holding current, showing that the GIRK1/GIRK4 channel isexpressed in this oocyte. Subsequent application of 100 mM GABAactivated a large inward current (middle panel). Negative and positivecontrol experiments are shown from oocytes expressing the GABA_(B)-R2receptor alone (left panel) and those expressing the adenosine A1receptor (right panel).

[0055]FIG. 13. Current-voltage curves in an oocyte expressingGABA_(B)-R1, GABA_(B)-R2 and the potassium channels GIRK1 and GIRK4.

[0056] Current-voltage curves are shown for a single oocyte followingapplication of 200 ms voltage-clamp pulses from a holding potential of60 mV to test potentials between −100 mV and +50 mV. Steady-statecurrent is plotted against test potential in ND96 solution (lowpotassium), 90K solution (90 mM potassium) and 90K plus 100 mM GABA.Note the basal GIRK1/4 current recorded in 90K solution and the largeagonist-evoked activation of the GIRK potassium channel.

[0057]FIG. 14. GABA-mediated stimulation of [³⁵S]GTPγS binding activityis dependent on the relative levels of expression of GABA_(B)-R1 andGABA_(B)-R2 receptors

[0058] HEK293T cells were transfected with HA-GABA_(B)-R2 (1 μg) andG_(o1)α (1 μg) together with various amounts (0-1 μg) ofHA-GABA_(B)-R1b. Cells were harvested 48 h after transfection and P2membrane fractions were prepared. (A) Agonist stimulation of [³⁵S]GTPγSbinding activity measured in transfected cell membranes in the presenceof GABA (10 mM). Data are shown as stimulation above basal (cpm) and arethe mean±S.D. of triplicate measurements. (B) Cell membranes wereimmunoblotted with anti-HA antiserum to allow the relative levels ofHA-GABA_(B)-R2 and HA-GABA_(B)-R1b receptors to be evaluated.

[0059]FIG. 15. Co-expression of GABA_(B)-R1 and GABA_(B)-R2 receptors inHEK293T cells generates a high affinity GABA_(B) binding site similar tobrain GABA_(B) receptors.

[0060] P2 membrane fractions were prepared from HEK 293T cellstransfected using the same conditions described for GTPγS bindingstudies. % specific binding was determined for the displacement of[3H]-CGP54626 by GABA. Data shown are the mean of minimum of triplicatestudies±sem.

DETAILED DESCRIPTION OF THE INVENTION

[0061] Throughout the present specification and the accompanying claimsthe words “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

[0062] As previously explained, the present invention includes a numberof important aspects. In particular the present invention relates toisolated GABA_(B)-R2 receptor proteins and variants thereof, isolatedGABA_(B)-R1c receptor proteins and variants thereof, GABA_(B) receptorscomprising an heterodimer between a GABA_(B)-R1 receptor protein or avariant thereof and a GABA_(B)-R2 receptor protein or a variant thereof,as well as other related aspects. In the context of the presentinvention the wording “isolated” is intended to convey that the receptorprotein is not in its native state, insofar as it has been purified atleast to some extent or has been synthetically produced, for example byrecombinant methods. The term “isolated” therefore includes thepossibility of the receptor protein being in combination with otherbiological or non-biological material, such as cells, suspensions ofcells or cell fragments, proteins, peptides, organic or inorganicsolvents, or other materials where appropriate, but excludes thesituation where the receptor protein is in a state as found in nature.

[0063] Routine methods, as further explained in the subsequentexperimental section, can be employed to purify and/or synthesise thereceptor proteins according to the invention. Such methods are wellunderstood by persons skilled in the art, and include techniques such asthose disclosed in Sambrook, J. et al, 1989, the disclosure of which isincluded herein in its entirety by way of reference.

[0064] The present invention not only includes the GABA_(B) receptorproteins specifically recited, but also variants thereof. By the term“variant” what is meant throughout the specification and claims is thatother peptides or proteins which retain the same essential character ofthe receptor proteins for which sequence information is provided, arealso intended to be included within the scope of the invention. Forexample, other peptides or proteins with greater than about 80%,preferably at least 90% and particularly preferably at least 95%homology with the sequences provided are considered as variants of thereceptor proteins. Such variants may include the deletion, modificationor addition of single amino acids or groups of amino acids within theprotein sequence, as long as the biological functionality of the peptideis not adversely affected.

[0065] The invention also includes nucleotide sequences which encode forGABA_(B)-R2 or GABA_(B)-R1c receptors or variants thereof as well asnucleotide sequences which are complementary thereto. Preferably thenucleotide sequence is a DNA sequence and most preferably, a cDNAsequence.

[0066] The present invention also includes expression vectors whichcomprise nucleotide sequences encoding for the GABA_(B)-R2 orGABA_(B)-R1c receptor subtypes or variants thereof. A further aspect ofthe invention relates to an expression vector comprising nucleotidesequences encoding for a GABA_(B)-R1 receptor protein and a GABA_(B)-R2receptor protein or variants thereof. Such expression vectors areroutinely constructed in the art of molecular biology and may involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, which may be necessary, and which are positioned inthe correct orientation, in order to allow for protein expression.

[0067] The invention also includes cell lines which have been modifiedto express the novel receptor. Such cell lines include transient, orpreferably stable higher eukaryotic cell lines, such as mammalian cellsor insect cells, lower eukaryotic cells, such as yeast or prokaryoticcells such as bacterial cells. Particular examples of cells which havebeen modified by insertion of vectors encoding for the receptor proteinsaccording to the invention include HEK293T cells and oocytes. Preferablythe cell line selected will be one which is not only stable, but alsoallows for mature glycosylation and cell surface expression of theinventive receptors. In the case of the functional GABA_(B) receptorwhich comprises a heterodimer of GABA_(B)-R1 and GABA_(B)-R2 subunits,the cell line may include a single vector which allows for expression ofboth of the receptor subtypes, or alternatively separate vectors foreach subunit. It is preferred however, that the receptor subtypes shouldbe co-expressed in order to optimise the dimerisation process, whichwill result in full glycosylation and transport of the glycosylateddimer to the cell surface.

[0068] It is also possible for the receptors of the invention to betransiently expressed in a cell line or on a membrane, such as forexample in a baculovirus expression system. Such systems, which areadapted to express the receptors according to the invention, are alsoincluded within the scope of the present invention.

[0069] A particularly preferred aspect of the invention is theheterodimer formed between the GABA_(B)-R1 and GABA_(B)-R2 receptorproteins which results in the formation of a functional GABA₉ receptor.Without wishing to be bound by theory, it appears that the formation ofthe heterodimer takes place via the coiled-coil domains within thereceptor C-terminal tails, and that this in turn is a pre-requisite fortransport and full glycosylation of a GABA_(B)-R1, and also forgeneration of an high affinity GABA_(B) receptor at the cell surface.

[0070] The heterodimer which forms a functional GABA_(B) receptor cancomprise any GABA_(B)-R1 receptor subtype or splice variant, or variantsthereof. Although we are presently only aware of only one GABA_(B)-R2subtype, it is envisaged that the heterodimers according to the presentinvention can include other GABA_(B)-R2 subtypes or splice variantswhich have not yet been identified, as well as variants of the alreadyidentified GABA_(B)-R2 receptor proteins.

[0071] In particular, the functional GABA_(B) receptor may includeGABA_(B)-R1 receptor proteins selected from GABA_(B)-R1a, GABA_(B)-R1b,GABA_(B)-R1c splice variants, variants thereof or even other GABA_(B)-R1receptor subtypes or splice variants which have not yet been identified.

[0072] According to another aspect, the present invention also relatesto antibodies which have been raised by standard techniques and arespecific for the receptor proteins or variants thereof according to theinvention. Such antibodies could for example, be useful in purification,isolation or screening involving immuno precipitation techniques and maybe used as tools to further ellucidate GABA_(B) receptor function, orindeed as therapeutic agents in their own right. Antibodies may also beraised against specific epitopes of the receptors according to theinvention, as opposed to the monomer subunits.

[0073] An important aspect of the present invention is the use ofreceptor proteins according to the invention, particularly theheterodimer GABA_(B) receptor, in screening methods designed to identifycompounds which act as receptor ligands and which may be useful tomodulate receptor activity. In general terms, such screening methodswill involve contacting the receptor protein concerned, preferably theheterodimeric GABA_(B) receptor, with a test compound and then detectingmodulation in the receptor activity, or indeed detecting receptorinactivity, which results. The present invention also includes withinits scope those compounds which are identified as possessing usefulGABA_(B) receptor modulation activity, by the screening methods referredto above. The screening methods comprehended by the invention aregenerally well known to persons skilled in the art, and are furtherdiscussed in the experimental section which follows.

[0074] Another aspect of the present invention is the use of compoundswhich have been identified by screening techniques referred to above inthe treatment or prophylaxis of disorders which are responsive tomodulation of a GABA_(B) receptor activity, in a mammal. By the term“modulation” what is meant is that there will be either agonism orantagonism at the receptor site which results from ligand binding of thecompound at the receptor. GABA_(B) receptors have been implicated indisorders of the central nervous system (CNS), gastrointestinal (GI)tract, lungs and bladder and therefore modulation of GABA_(B) receptoractivity in these tissues will result in a positive therapeutic outcomein relation to such disorders. In particular, the compounds which willbe identified using the screening techniques according to the inventionwill have utility for treatment and/or prophylaxis of disorders such asspasticity, epilepsy, Alzheimer's disease, pain as well as affectivedisorders and feeding disorders. It is to be understood however, thatthe mention of such disorders is by way of example only, and is notintended to be limiting on the scope of the invention.

[0075] The compounds which are identified according to the screeningmethods outlined above may be formulated with standard pharmaceuticallyacceptable carriers and/or excipients as is routine in thepharmaceutical art, and as fully described in Remmington'sPharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania,17th Ed, 1985, the disclosure of which is included herein in itsentirety by way of reference.

[0076] The compounds may be administered via enteral or parenteralroutes such as via oral, buccal, anal, pulmonary, intravenous,intraarterial, intramuscular, intraperitoneal, topical or otherappropriate administration routes.

[0077] Other aspects of the present invention will be further explained,by way of example, in the appended experimental section.

[0078] Experimental

[0079] Results

[0080] 1. Cloning of Human GABA_(B)-R1 and a novel Receptor subtype,GABA_(B)-R2 Human homologues to the rat GABA_(B)-R1a and 1b splicevariants were identified from ESTs and subcloned from Human cerebellumcDNA, using a combination of PCR and Rapid amplification of cDNA ends(RACE) PCR. Human GABA_(B)-R1a and 1b sequences reveal over 99% identityto the rat GABA_(B)-R1a and GABA_(B)-R1b (data not shown). Thesereceptors, like their rat counterparts, both have signal sequences,followed by extended N-termini, a typical seven-transmembrane topologyand short intracellular C-terminal tail. The N-terminus encodes the GABAbinding domain, which is predicted by limited homology to bacterialperiplasmic proteins to exist as two globular domains that capture GABA(Bettler et al., 1998), as well as three potential N-glycosylationsites. Interestingly the GABA_(B)-R1a splice variant N-terminus encodes129 amino acids over that of GABA_(B)-R1b, which encode two tandemcopies of the ‘short consensus repeat’ or sushi domain. Sushi domainsare approximately 60 amino acids in length and exist in a wide range ofproteins involved in complement and cell-cell adhesion (Chou andHeinrikson, 1997). Therefore the sushi domains within GABA_(B)-R1a maydirect protein-protein interactions, possibly through cell-cell contactand may reflect a further role for GABA_(B)-R1a, over and above that ofGABA_(B)-R1b. Interestingly during the isolation of these clones, anovel N-terminal splice variant, GABA_(B)-R1c was identified.GABA_(B)-R1c differs from GABA_(B)-R1a by a 185 bp deletion from bases290 to 475 (see FIG. 2). This region encodes one of the two Sushidomains unique to GABA.-R1a and therefore the GABA_(B)-R1a andGABA_(B)-R1c splice variants, together with their cellular localisation,may be significant in the biology of GABA_(B) receptors. Indeed, in situhybridisations suggest that GABA_(B)-R1a and GABA_(B)-R1b have differentsub-cellular localisations, with GABA_(B)-R1a expressed at pre-synapticrather than at post-synaptic sites (Bettler et al., 1998).

[0081] Database searches also identified a number of ESTs showing weakerhomology to GABA_(B)-R1, suggesting the existence of a novel GABA_(B)receptor subtype. Using PCR on Human Brain cerebellum cDNA, we confirmedthe existence of such a novel GABA_(B) receptor which we cloned andsequenced (FIG. 1). This novel receptor, which we have calledGABA_(B)-R2, shows an overall 54% similarity and 35% identity toGABA_(B)-R1 over the full length of the protein (FIG. 2). As expected,hydrophobicity profiles for GABA_(B)-R2 (FIG. 3) suggested that theprotein has a 42 amino acid signal peptide followed by an extracellularN-terminal domain comparable in size to that of GABA_(B)-R1b and sevenmembrane spanning regions. In total five N-glycosylation sites werepredicted over the N-terminal domain, three of which are conservedwithin GABA_(B)-R1. Finally, the receptor encodes an intracellularC-terminal domain, which is considerably larger than that ofGABA_(B)-R1. No sushi domains were identified within GABA_(B)-R2sequence and we have no evidence for any splice variants to date.

[0082] 2. Tissue Distribution

[0083] Expression levels of both GABA_(B)-R1 and GABA_(B)-R2 weredetermined and compared in different tissues and developmental stages byprobing Human RNA Master Blots (Clontech). These blots contain polyA⁺RNA samples from 50 human tissues that have been normalized to the mRNAexpression levels of eight different “housekeeping” genes. GABA_(B)-R1levels were examined using a pan-specific probe covering all splicevariants (FIG. 4a) and the blots indicate that in accordance with theobservations of Kaupmann et al., (1997), GABA_(B)-R1 is highly expressedin the CNS, in all areas of the brain and spinal cord. However, incontrast to Kaupmann et al., (1997), we find that GABA_(B)-R1 is alsoexpressed at comparable levels in peripheral tissues, with particularlyhigh levels of expression in the pituitary, lung, ovary, kidney, smallintestine, and spleen. In marked contrast, GABA_(B)-R2 is specificallyexpressed at high levels only in the CNS, with the possible exception ofspinal cord where expression appears somewhat lower. No signal is seenfor peripheral tissues, in either adult or fetal tissues (FIG. 4b). Thismarkedly different distribution of mRNA levels between GABA_(B)-R1 andGABA_(B)-R2 suggests that the two subtypes may have distinct roles inthe CNS and periphery.

[0084] 3. Initial Expression Studies

[0085] We reasoned that GABA_(B)-R2 could be a high affinity GABA_(B)receptor and therefore, expressed the receptor in both Xenopus oocytesand HEK293T cells and looked for functional responses. However, despiterepeated attempts, we were unable to detect any functional activation ofGABA_(B)-R2 or indeed, GABA_(B)-R1a, GABA₆-R1b or GABA_(B)-R1c receptorsby either GABA itself or GABA_(B) selective agonists (See FIGS. 9, 11and 12). Several lines of evidence clearly indicated that GABA_(B)-R1was not expressed as predicted in vivo. Firstly, flow cytometry ofHEK293T cells, expressing GABA_(B)-R1b, revealed that receptors wereretained on internal membranes rather than expressed at the cell surface(FIG. 7). Secondly, GABA_(B)-R1a and GABA_(B)-R1b were expressed asimmature glycoproteins, by virtue of their sensitivity toendoglycosidases F and H (FIG. 8, lanes 1-6) and finally, GABA_(B)-R1co-expression in oocytes with either GIRK or CFTR, gave no indication ofa functional response (data not shown). We concluded that someadditional co-factor must be required to promote a functional response.

[0086] 4. Yeast Two Hybrid Library Screening

[0087] The calcitonin-receptor like receptor is retained as an immatureglycoprotein within the endoplasmic reticulum and requires an accessoryprotein from the recently identified RAMP protein family to transportthe receptor to the surface to generate a functional CGRP (Calcitoningene-related peptide) or adrenomedullin receptor (McLatchie et al.,1998). We anticipated that GABA_(B)-R1 receptors should require ananalogous trafficking factor or some other protein co-factor for itstransport to the cell surface to generate a high affinity receptor. Toidentify such potential interacting proteins, a yeast two hybrid libraryscreen was run using the C-terminal 108 amino acids of GABA_(B)-R1against a Human Brain cDNA library. Interestingly, motif searchesrevealed a strong coiled-coil domain within these 108 residues, astructure known to mediate protein-protein interactions (Lupas, 1996).From a total of 4.3×10⁶ cDNAs, 122 positives hits were recovered, 33 ofwhich encoded the whole C-terminal domain of GABA_(B)-R2. This domain ofthe GABA_(B)-R2 is likewise predicted to contain a coiled-coil motif,which aligns exactly with that of GABA_(B)-R1 (see FIG. 2). Thisobservation strongly suggests that the two receptors interact via theirC-termini to form a heterodimer. Significantly, the screen did notretrieve the C-terminal domain of the GABA_(B)-R1 itself, implying thatGABA_(B)-R1 is unable to homodimerise. This interaction was testeddirectly in the yeast two hybrid system using the C-termini of the tworeceptors (FIG. 5). GABA_(B)-R1 and GABA_(B)-R2 were able to stronglyinteract via their C-termini, whilst neither receptor was able tohomodimerise. This observation suggested that GABA_(B)-R1 andGABA_(B)-R2 form heterodimers via their C-terminal coiled-coil domainsand led to speculation that homodimerisation may bring about afunctional binding site in vivo. Therefore, we next confirmed theinteraction between the two receptor subtypes by immunoprecipitationstudies upon whole epitope-tagged receptor in transfected HEK293T cells.

[0088] 5. Co-Immunoprecipitation Studies.

[0089] Epitope tagged receptors, Myc-GABA_(B)-R1b and HA-GABA_(B)-R2were transiently expressed in HEK293T cells either alone or incombination. Immunoprecipitation of Myc-GABA_(B)-R1b fromdetergent-solubilised cell fractions with Myc antisera led toimmunodetection of HA-GABA_(B)-R2 within immune complexes using HA asthe primary antibody, but only upon receptor co-expression (FIG. 6,lanes 1-3). GABA_(B)-R1 and GABA_(B)-R2 association was confirmed byco-immunodetection of Myc-GABA_(B)-R1b from immune complexes capturedusing the anti-HA antibody. Once again, co-immunoprecipitation couldonly be seen when the two receptor forms were co-expressed (FIG. 6,lanes 4-6). Hence in agreement with the yeast two hybrid observations,these data provide compelling evidence for heterodimerisation betweenfull-length expressed GABA_(B)-R1 and GABA_(B)-R2 in mammalian cells.Therefore, we next examined GABA_(B) receptor responses followingco-expression of both receptor subtypes in HEK293T cells or in Xenopusoocytes.

[0090] 6. Surface Expression of the Heterodimer

[0091] HEK293T cells were transiently transfected with Myc-GABA_(B)-R1balone or in combination with HA-GABA_(B)-R2 and transfectants analysedby flow cytometry (FIG. 7). Myc-immunoreactivity could not be detectedon the surface of cells transfected with Myc-GABA_(B)-R1b alone (FIG.7a), although cell permeabilisation revealed immunoreactivity in 35%(n=3) of the cell population (FIG. 7b). This latter observationindicated that cells were efficiently transfected and suggested thatexpressed Myc-GABA_(B)-R1 receptors were localised exclusively oninternal membranes. In contrast, 14% (n=3) of HEK293T cells transfectedwith HA-GABA %-R2 showed surface immunoreactivity (FIG. 7c). However,co-transfection of both Myc-GABA_(B)-R1b and HA-GABA_(B)-R2 led to theappearance of Myc-GABA_(B)-R1b on the surface of 20% (n=3) of cellsanalysed (FIG. 7a), strongly suggesting that co-expression ofGABA_(B)-R1b with GABA_(B)-R2 is necessary for surface expression ofGABA_(B)-R1b.

[0092] 7. Receptor Glycosylation Studies

[0093] Endoglycosidases F and H can be used to differentiate betweencore and terminally glycosylated N-linked glycoproteins. Therefore,these enzymes were used to examine the glycosylation status of bothGABA_(B)-R1 and GABA_(B)-R2 following expression in HEK293T cells.Membranes from transfected cells were treated with eitherendoglycosidase F or endoglycosidase H and expressed GABA_(B) receptorswere characterised by immunoblotting to compare relative electrophoreticmobilities of the receptors (FIG. 8). Cell membranes expressing eitherGABA_(B)-R1a or 1b produced distinct bands of M_(r) 130 and 100Krespectively (FIG. 8, lanes 1 and 4) which following endoglycosidase Ftreatment, decreased in size to single immunoreactive species of M_(r)110 and 80K; representing GABA_(B)-R1a and GABA_(B)-R1b respectively(FIG. 8, lanes 2 and 5). This shows that recombinant GABA_(B)-R1a and 1bare glycoproteins, in agreement with the observations of Kaupmann etal., (1997). However, both GABA_(B)-R1a and 1b splice variant forms werealso sensitive to endoglycosidase H treatment, indicating that theexpressed proteins are only core glycosylated (lanes 3 and 6) and lackterminal glycosylation. This observation, together with the FACSanalysis, suggests that the proteins are immaturely glycosylated andretained on internal membranes. Significantly, when either GABA_(B)-R1a(lanes 7-9) or GABA_(B)-R1b (lanes 10-12) was co-expressed withHA-GABA_(B)-R2, a component of GABA_(B)-R1a or 1b was resistant toendoglycosidase H digestion suggesting that when co-expressed withGABA_(B)-R2, a significant fraction of GABA_(B)-R1 is now a matureglycoprotein (lanes 9 and 12).

[0094] Similar studies with HA-GABA_(B)-R2 gave an immunoreactivespecies with an M_(r) of 120 K (FIG. 8, lanes 13, 16, 19) which wassensitive to endoglycosidase F (lanes 14, 17 and 20) but resistant toendoglycosidase H (lanes 15, 18 and 21) treatment, whether expressedalone or in combination with GABA_(B)-R1. Thus, these data indicate thatexpressed HA-GABA_(B)-R2 is a mature glycoprotein whose glycosylationstatus is not affected by co-expression with GABA_(B)-R1. Thus,heterodimerisation between GABA_(B)-R1 and GABA_(B)-R2, possibly in theGolgi complex, could be a prerequisite for maturation and transport ofGABA_(B)-R1 to the plasma membrane.

[0095] 8. Functional Studies

[0096] To determine whether co-expression of GABA_(B)-R1 and GABA_(B)-R2and its subsequent mature glycosylation and cell surface expression,generated a receptor complex able to functionally respond to GABA, wemeasured three types of signalling. We used transiently transfectedHEK239T cells to examine firstly, activation of [³⁵S]GTPγS binding inmembranes and secondly, inhibition of forskolin stimulated cAMPactivation in whole cells. Thirdly we expressed GABA_(B)-R1 andGABA_(B)-R2 in Xenopus oocytes, expressing either the cystic fibrosistransmembrane regulator (CFTR) or inwardly rectifying K⁺ channels (GIRKand KATP) and examined activation of ion flux in response to agonist.

[0097] i. [³⁵S]GTPγS Binding

[0098] No GABA stimulated [³⁵S]GTPγS binding was observed in membranesprepared from cells transfected with either GABA_(B)-R1 orHA-GABA_(B)-R2 in combination with G_(o1)α. However, co-expression ofGABA_(B)-R1 and HA-GABA_(B)-R2 together with G_(o1)α resulted in arobust stimulation of [³⁵S]GTPγS binding activity (FIG. 9a). This wasfound to be concentration-dependent with similar EC₅₀ (mean,±S.E.M.,n=3) values determined for membranes from cells transfected withHA-GABA_(B)-R2 and G_(o1)α together with either GABA_(B)-R1a(9.5+1.1×10⁻⁵M) or GABA_(B)-R1b (7.8±0.4×10⁻⁵M) (FIG. 9b). These valuesare equivalent to those of GABA-mediated stimulation of [³⁵S]GTPγSbinding to rat brain membranes (5.9±0.4×10⁻⁵M) (data not shown). We wereconcerned that an N-terminal HA epitope tag on GABA_(B)-R2 could alterreceptor function and so we performed parallel studies in HEK293T cells,expressing untagged versions of GABA_(B)-R2 and GABA_(B)-R1 togetherwith G_(o1)α. Similar efficacies and potencies of GABA action wereobserved in membranes from these cells, as reported for the epitopetagged receptors (data not shown), clearly suggesting that the additionof these peptide sequences to the N-termini of GABA_(B)-R2 andGABA_(B)-R1 did not significantly alter receptor function. It isnoteworthy that a measurable GABA-mediated elevation of [³⁵S]GTPγSbinding activity was only observed upon co-expression of GABA_(B)-R1 andHA-GABA_(B)-R2 together with additional G_(o1)α (FIG. 10). Therequirement for additional G protein is most likely due to relativelylow levels of endogenously expressed G_(i/o) family G proteins, thusprecluding a discernible GABA-mediated response upon GABA_(B)-R1 andGABA_(B)-R2 co-expression.

[0099] ii cAMP Inhibition

[0100] Similar results were obtained from HEK293T cells transientlytransfected with GABA_(B)-R1 and GABA_(B)-R2, using inhibition offorskolin evoked cAMP as a readout. Once again, functional responseswere only observed when both GABA_(B)-R1 and GABA_(B)-R2 wereco-expressed (FIG. 11).

[0101] iii Xenopus Oocytes

[0102] Xenopus oocytes can assay for three classes of G-protein:

[0103] 1) Endogenous oocyte Ca²⁺-activated chloride conductance canassay for activation of G_(q) and a subsequent rise in intracellularcalcium (Uezono et al., 1993).

[0104] 2) Cystic fibrosis transmembrane regulator (CFTR), which containsa cAMP-activated chloride channel, can assay for receptor activation viaG_(s) or G_(i/o) (Uezono et al., 1993; Wotta et al., 1997).

[0105] 3) G-protein regulated potassium channels GIRK1 (Kir 3.1; Kubo etal., 1993) and GIRK4 (or CIR, Kir 3.4, Kaprivinsky et al., 1995),injected in equal amounts to generate a heteromeric channel, can assayfor activation of pertussis toxin sensitive G-proteins (Kovoor et al.,1997).

[0106] No functional responses to GABA or baclofen were seen when clonedGABA_(B)-R1a, GABA_(B)-R1b or GABA_(B)-R2 receptors were expressed inoocytes in combination with CFTR or GIRK1/4 (data not shown; see FIG.12b). When GABA_(B)-R1 and GABA_(B)-R2 were co-expressed with CFTR,several significant, robust responses were recorded followingapplication of 100 μM GABA (FIG. 12a). Moreover, repeated application ofGABA led to a progressive increase in the size of the CFTR response,suggesting that the functional response of the heterodimer is nowsensitised to further challenge by agonist. This phenomenon has not beenobserved for other cloned receptors expressed in oocytes and may berelated to the heterodimerisation or even oligomerisation of theGABA_(B) receptors. Finally, two other GABA_(B)-selective agonists,Baclofen and SKF97541 elicted similar functional responses through CFTRto that of GABA (FIG. 12a). In contrast, antagonists gave no response(data not shown).

[0107] Next, we examined the GABA_(B)-R1/GABA_(B)-R2 heterodimer withthe G-protein regulated potassium channels GIRK1 and GIRK4 and onceagain found agonist dependant responses. Time course plots were examinedfor three individual oocytes expressing GABA_(B)-R2 alone (left panel),GABA_(B)-R1 plus GABA_(B)-R2 (middle panel) and the adenosine A1receptor (as a positive control, right panel) (FIG. 12b). In each case,switching from a low potassium physiological solution (ND96) to a highpotassium extracellular solution (90 mM K⁺) led to an inward shift inholding current, resulting from agonist-independent influx of potassiumions through the GIRK1/4 channel. No GABA response was seen in oocytesexpressing GABA_(B)-R2 in isolation (FIG. 12b, left panel) andsimilarly, GABA_(B)-R1a and GABA_(B)-R1b expressed alone also gave noresponse to GABA (data not shown). Significantly, a large GABA responsewas recorded in oocytes co-expressing GABA_(B)-R1 and GABA_(B)-R2 (FIG.12b, middle panel) of a similar magnitude to that of the adenosine A1receptor in response to the agonist NECA (FIG. 12b, right panel). Thus,once again co-expression of the two receptor subtypes elicits afunctional agonist-dependant response, whereas expression of eithersubtype receptor alone does not. We also examined whether co-expressionof the two receptors in oocytes could activate endogenous Ca²+-activatedchloride conductance. No evidence for activation was seen (data notshown) suggesting that at least in oocytes, the GABA_(B)-R1/GABA_(B)-R2receptor complex does not signal through G_(q). Finally, acurrent-voltage curve were constructed for an oocyte co-expressingGABA_(B)-R1 and GABA_(B)-R2 (FIG. 13). This clearly demonstrates thatGABA, bound to the GABA_(B) receptor, activates a large inwardlyrectifying current consistent with activation of the GIRK potassiumchannel in a fully dose dependant manner.

[0108] 9. Stoichiometric Studies on the Heterodimer

[0109] Since co-expression of GABA_(B)-R1 and GABA_(B)-R2 is necessaryfor a functional GABA_(B) receptor, we decided to investigatestoichiometric ratio between the two receptor subtypes in vivo. Relativelevels of expression for both GABA_(B)-R1 and GABA_(B)-R2 were measuredfollowing transfection into HEK293T cells and compared to receptorfunction, as determined by GTPγS binding (FIG. 14). Increasing amountsof HA-GABA_(B)-R1 (up to 1 μg) plasmid were transfected into HEK293Tcells along with a constant (1 μg) amount of HA-GABA_(B)-R2. GABA causedstimulation of [³⁵S]GTPγS binding above basal levels in membranesextracted from these cells, which increased with increasing amount oftransfected HA-GABA_(B)-R1 until binding reached a plateau when levelsof HA-GABA_(B)-R1 were greater than 0.25 μg (FIG. 14a). Immunoblottingof the same membrane samples revealed equivalent levels of expression ofHA-GABA_(B)-R1 and HA-GABA_(B)-R2 in membranes transfected with 0.25-0.5μg of HA-GABA_(B)-R1 (FIG. 14b). This corresponded to the plateau ofGABA-mediated elevation of [³⁵S]GTPγS binding activity and thereforestrongly suggests that GABA_(B)-R1 and GABA_(B)-R2 functionally interactin a 1:1 stoichiometric ratio.

[0110] 10. Competition Binding Studies

[0111] Finally, we determined whether the observed functional responseswere due to a high affinity GABA_(B) receptor, composed of a heterodimerof the two receptors. HEK293T cells were transfected with either 1 μgHA-GABA_(B)-R1b and HA-GABA_(B)-R2 individually or with increasingamounts (up to 1 μg) of HA-GABA_(B)-R1b and a fixed amount (1 μg) ofHA-GABA_(B)-R2 together with G_(o1)α. Competition binding assays werethen performed upon purified membranes. Expression of HA-GABA_(B)-R1balone produced high levels of specific binding of [³H]-CGP54626(Bittiger et al., 1992), a structural analogue of [¹²⁵I]-CGP64213 andthe antagonist originally used to expression clone GABA_(B)-R1 (Kaupmannet al., 1997). However, as previously reported for [¹²⁵I]-CGP64213, GABAinhibition curves were significantly shifted to the right compared withbinding to rat brain membranes (FIG. 15), giving approximately 22-foldlower IC50 than rat brain binding. Significantly, co-expression ofequivalent amounts of HA-GABA_(B)-R1b and HA-GABA_(B)-R2 proteinrevealed high levels of specific binding. In a control experiment usinguntagged receptors similar values were obtained (data not shown).Achievement of a 1:1 stoichiometric ratio of expression ofHA-GABA_(B)-R1b and HA-GABA_(B)-R2 led to agonist inhibition curvessimilar to those obtained in rat brain membranes (IC50±95% confidenceintervals for 1 μg HA-GABA_(B)-R2/0.25 kg HA-GABA_(B)-R1b=2.29 μM(1.48-3.55 μM) and for rat brain=1.04 μM (0.69-1.58 μM). Such comparablelevels of receptor expression were also shown to permit optimal agonistactivation in the GTPγS assay (see FIG. 14). Alteration of receptorratio from 1:1, such that GABA_(B)-R1b was the most prevalent receptor,led to reduced agonist affinity, presumably due to binding atnon-dimerised and immaturely glycosylated GABA_(B)-R1b receptors (FIG.15).

[0112] In addition, despite its apparent cell surface expression, wewere unable to detect any [³H]-CGP54626 specific binding to HEK293Tcells transiently transfected with HA-GABA_(B)-R2 alone (data notshown). We conclude that heterodimerisation of the GABA_(B)-R1 andGABA_(B)-R2 subtypes are necessary to generate a high affinity GABA_(B)receptor. There are a number of possible explanations for the change inGABA affinity following co-expression of the two receptor subtypes.Appearance of the GABA_(B) receptor complex at the cell surface would beexpected to allow G protein coupling of the receptor which wouldincrease agonist affinity. However, in previous studies is has beenshown that the lack of G protein coupling alone cannot account for thedifference in agonist affinity between rat brain receptors andGABA_(B)-R1 (Kaupmann et al., 1997). Furthermore, we have noted that[³H]-CGP54626 appears to primarily bind the low affinity state of thereceptor, even in rat brain membranes, as demonstrated by the fact thatGTPγS is unable to shift agonist inhibition curves and actuallyincreases the level of ³H-CGP54626 specific binding (data not shown).Therefore, a more likely explanation for the change in GABA affinityfollowing co-expression of the two GABA_(B) receptors is thatheterodimerisation together with the mature glycosylation state of theprotein, produces a binding site conformation with an inherent higheraffinity.

[0113] Discussion

[0114] Functional GABA_(B) receptors within the CNS comprise a cellsurface heterodimer of two distinct 7-transmembrane receptor subunits,GABA_(B)-R1 and GABA_(B)-R2 in a 1:1 stoichiometric ratio. In vivo,GABA_(B) receptors may exist simply as heterodimers or form even largermultimeric complexes of many heterodimers. Formation of the heterodimervia the coiled-coil domains within the receptor C-terminal tails appearsto be a pre-requisite for transport and full glycosylation ofGABA_(B)-R1, as well as for the generation of a high affinity GABA_(B)receptor at the cell surface. Using this information, we have been ableto reproduce GABA_(B) sites in both mammalian HEK293T cells as well asin oocytes, using several functional readouts such as activation of ionflux through CFTR or GIRK in oocytes, or inhibition of adenylyl cyclasein HEK293T cells. Indeed the lack of functional responses in cellsexpressing GABA_(B)-R1 alone and the need for expression of a second 7TMreceptor explains why many groups have encountered extreme difficulty inexpression cloning a GABA_(B) receptor via conventional means. Webelieve this is the first report of receptor heterodimerisation as anobligate requirement to generate a high affinity, fully functionalreceptor in recombinant systems, which is fully equivalent to that ofendogenous tissues.

[0115] Dimerisation has been reported for other receptor families, suchas the opioid family as a part of their desensitisation process, theβ2-adrenergic receptor, where homodimers may play a role in signalling,and the metabotropic glutamate receptors (mGluRs, Hebert et al., 1996;Romano et al., 1996; Cvejic et al., 1997, Hebert and Bouvier, 1998).Significantly, dimerisation in these receptor families does not appearto be an absolute requirement for functional coupling in recombinantsystems. In the case of the mGluRs, which are a closely related receptorfamily to GABA_(B) (Kaupmann et al., 1997), homodimerisation is mediatedthrough disulphide bridges between the N-terminal extracellular domainsrather than a C-terminal coiled-oil. Indeed, heterodimerisation betweentwo 7-transmembrane receptors, leading to both trafficking and matureglycosylation of the proteins to yield a functional receptor isunprecedented and is unique in the GPCR field. Certainly, mGluRs havenot been found to form heterodimers (Romano et al., 1996) and the factthat two such closely related receptors families have evolved suchdifferent mechanisms of dimer formation suggests that this is afundamentally important process for receptor function.

[0116] In vivo, pharmacological evidence suggests that there are manydifferent GABA_(B) receptor subtypes, both within the CNS as well as inperipheral tissues. How are such pharmacological subtypes of GABA_(B)receptors formed? Only GABA_(B)-R1 and GABA_(B)-R2 have been identifiedas separate genes to date and database trawling has not identified anyfurther receptors homologous to known GABA_(B) receptors. This does notexclude the possibility that more, as yet unrecognised GABA_(B)receptors do exist. Differences in distribution exist for the twoGABA_(B) receptors, for example GABA_(B)-R2 is specifically expressed inthe CNS whereas GABA_(B)-R1 is expressed in both central and peripheralsites. These differences in distribution clearly add further complexityleading to the pharmacologically distinct receptor subtypes. Moreover,the genes encoding the GABA_(B) receptors may be differentially spliced.GABA_(B)-R1 encodes three N-terminal splice variants and yet more mayremain to be detected. Interestingly, these splice variants havealterations in their N-terminal extracellular domain, the regioninvolved in GABA binding (Takahashi et al., 1993, O'Hara et al., 1993)and encode either two (GABA_(B)-R1a), one (GABA_(B)-R1c) or no(GABA_(B)-R1b) sushi domains. Given that the sushi domains mediatecell-cell protein-protein contact, the differences in these three splicevariants may account for yet more of the pharmacologically definedGABA_(B) receptor subtypes. To date, we have not detected any splicevariants to GABA_(B)-R2. Furthermore there are significant differencesin the distribution of the individual splice variants suggesting thatthey may serve different functions within the CNS. For instance,GABA_(B)-R1a splice variant is reported as presynaptic within the brain(Bettler et al., 1998) and therefore may define presynaptic GABA_(B)autoreceptors. It seems likely that these splice variants of GABA_(B)-R1may account for at least some of the pharmacologically defined subtypes.Finally, with this novel observation of obligate receptorheterodimerisation, a further level of complexity has been added sincefunctional GABA_(B) binding sites require a heterodimerisation partner.

[0117] Now the molecular nature of the GABA_(B) receptor is more fullyunderstood, recombinant systems can be established for high throughputscreening for compounds against individual pharmacologically definedGABA_(B) sites. By these means, compounds with greater specificity andwith fewer unwanted side effects can be discovered. For this, GABA₉-R1and GABA_(B)-R2 (including all spice variants, and any fragments of thereceptor) should be co-expressed either stably or transiently insuitable host cells. Suitable host cells include higher eukaryotic celllines, such as mammalian cells, insect cells, lower eukaryotic cells,such as yeast or prokaryotic cells such as a bacterial cells. Screeningassays with these recombinant cell lines could involve the use ofradioligand binding to the dimer or individual subunits within thedimer. The activity profile in a binding assay to the dimer is likely tobe different from the activity of compounds assayed using binding assaysto GABA_(B)-RI alone due to alterations in the glycosylation status andthe conformation of the receptor as a result of co-expressingGABA_(B)-R1 or GABA_(B)-R2. Functional assays, which measure eventsdownstream of receptor activation, can also be used for screeningcompounds. Such assays include [S]-GTPγS binding to membranes isolatedfrom cells expressing the dimers activation or inhibition of ionchannels using electrophysiological recording or ion flux assays;mobilisation of intracellular calcium; modulation of cAMP levels;activation or inhibition of MAP kinase pathways or alterations in theactivity of transcription factors with the use of reporter genes.Further to this, secondary screens can be established in a similarmanner, using different heterodimer combinations to exclude unwantedactivity and thereby establish subtype selective GABA_(B) compounds.

[0118] In addition, any approach targetting the disruption orenhancemant of dimer formation of the GABA_(B) heterodimer couldrepresent a novel therapeutic approach with which to target GABA_(B)receptors. Such strategies could include peptides or proteins physicallyassociated with the coiled-coil domain or indeed, any other interactingregions of the dimer. Small molecules could also be identified which actat the points of contact formed by interaction of the components of thedimer. These may either promote or enhance the receptor function.Finally, antibodies could be made which specifically recognise epitopeson the dimer, as opposed to the monomer subunits. These could be used astools to further elucidate the function of GABA_(B) receptors in diseaseor as therapeutic agents in their own right.

[0119] Methods

[0120] DNA Manipulation

[0121] Standard molecular biology protocols were used throughout(Sambrook et al., 1989) and all bacterial manipulations used Escherichiacoli XL-1 Blue (Stratagene) according to the manufacturers instructions.Standard PCR conditions were used throughout, unless otherwise stated.PCR reaction mixture contained 10-50 ng of target DNA, 1 pmol of eachprimer; 200 μM dNTPs and 2.5U of either Taq polymerase (Perkin-Elmer) orPful polymerase (Stratagene) with the appropriate buffer as supplied bythe manufacturer. Cycling parameters were 1 cycle 95° C. 2 mins; 25cycles 95° C. 45 secs 55° C. 45 secs 72° C. 1 min; 1 cycle 72° C. 10mins. All PCR were carried out using either a Perkin Elmer 9600 PCRmachine or a Robocycler Gradient 96 (Stratagene) PCR machine.

[0122] GABA_(B)-R1-Cloning of Human homologues and Splice Variants

[0123] Several human EST's (X90542; X90543; D80024; M348199; T0671 1;T07518 and AA38224) were identified as homologous to the ratGABA_(B)-R1a and GABA_(B)-R1b sequences (Y10369; Y10370). The ESTs werealigned and the predicted open reading frame was amplified by RT-PCRfrom human brain cerebellum polyA⁺ RNA (Clontech) using the SuperscriptPreamplification System (Life Technologies). The 3′ end of the receptor(1545-2538 bp; GABA_(B)-R1b) was amplified using primers5′-GCGACTGCTGTGGGCTGCTTACT GGC-3 and5′-GCGAATTCCCTGTCCTCCCTCACCCTACCC-3′. The central section (277-1737 bpof GABA_(B)-R1b) was amplified using 5′-CCGAGCTCMGCTCATCCACCACG-3′ and5′-TCTTCCTCCACTCCTTCTTTTCTT-3′. PCR products were subcloned intopCR-Script SK(+) (PCR-script Amp cloning kit; Stratagene). Error freePCR product were assembled in a three-way BstEII, SacI and EcoRIligation and subcloned into pBluescript SK (−) (Stratagene).

[0124] The N-termini of the splice variants were generated using RACE(rapid amplification of cDNA ends) PCR with the Marathon cDNAamplification kit against Marathon-Ready human cerebellum cDNA(Clontech). RACE PCR was primed from a conserved sequence withinGABA_(B)-R1 using primer 5′-TGAGCTGGAGCCATAGGAAAGCACMT-3′ to generate a700 bp product. This further PCR amplified using the AP2 primer(Marathon) and a second internal GABA_(B)-R1 primer5′-GATCTTGATAGGGTCGTTGTAGAGCA-3′. The resulting 600 bp product wassubcloned using the Zero blunt PCR cloning kit (Invitrogen). Sequenceinformation achieved from this RACE PCR was used to clone the N-terminusof the GABA_(B)-R1b splice variant, using primers5′--GCTCCTAACGCTCCCCAACA-3′ and 5′-GGCCTGGATCACACTTGCTG-3′ intopCR-Script SK (+)(Stratagene). Human GABA_(B)-R1a 5′ sequences wereretrieved from Incyte database EST's (1005101;3289832) and used todesign primers 5′-CCCMCGCCACCTCAGAAG-3′ and 5′-CCGCTCATGGGAAACAGTG C-3′.PCR on cerebellum cDNA and KELLY neuroblastoma cell line cDNA producedtwo discreet bands at 300 bp and 400 bp, which were cloned intopCR-Script SK (+) (Stratagene). Sequencing revealed that the 400 bpproduct encoded some of the Human GABA_(B)R1a 5′ sequences and the 300bp product encoded the novel splice variant, GABA_(B)-R1c. Next, primer,5′-CCCCGGCACACATACTCAATCTCATAG-3′ was designed to RACE PCR the missing˜225 bp of GABA_(B)-R1a. A 250 bp product was obtained and reamplifledusing primer 5′-CCGGTACCTGATGCCCCCTTCC-3′ with primer AP2 (Marathon). A˜250 bp band was once again generated, subcloned into pCR-Script SK (+)and when sequenced, encoded the 5′ end of GABA_(B)-R1a. Next, clonesspanning both the conserved receptor sequence and the %′ ends of thesplice variants GABA_(B)-R1a and GABA_(B)-R1c were generated. Primer5′-CGAGATGTTGCTGCTGCTGCTA-3′, priming from the start codon and thereverse RACE primer generated a predicted ˜800 bp band and this wassubcloned into pCR-Script SK(+). Now, full-length GABA_(B)-R1a,GABA_(B)-R1b and GABA_(B)-R1c clones can be assembled in pcDNA3.1(−)(Invitrogen). For GABA_(B)-R1b, 5′ sequences, restricted NotI/SacI, andthe conserved region of the receptor, cut EcoRI/SacI were bothco-ligated into pcDNA3.1 (−), restricted NotI/EcoRI. Likewise, theGABA₈-R1a and GABA₉-R1c 5′ fragments were subcloned XhoI/SacI with theEcoRI/SacI conserved fragment and co-ligated into pcDNA3.1(−), cutXhoI/EcoRI to reconstitute full length clones.

[0125] Tagging of GABA_(B)-R1b

[0126] GABA_(B)-R1b was tagged with either myc or HA epitopes. PCRprimers 5′-TAGGATCCCACTCCCCCCATCCC-3′ and 5′-CCAGCGTGGAGACAGAGCTG-3′were used to amplify a region immediately following the proposed signalsequence (position 88) to approx. 20 bp downstream of a unique PstI siteat position 389 of the coding sequence, creating a unique 5′ in-frameBamHI site. This fragment was cloned, BamHI/PstI, into a vectorcontaining the CD97 signal sequence, the myc epitope and an in-frameBamHI site. This construct also contains a NotI site 5′ to the CD97signal sequence and an EcoRI site downstream of the PstI site.GABA_(B)-R1b sequences downstream to the PstI site and upto an externalEcoRI site were subcloned from full length receptor into the vectordescribed above likewise cut with PstI/EcoRI, to assemble full lengthtagged GABA_(B)-R1b. CD97 signal sequence, myc epitope and GABA_(B)-R1bcoding sequence were subcloned, NotI/EcoRI, into pCDNA3.1(−)(Invitrogen). HA epitope was added to GABA_(B)-R1b by co-ligation of the5′ BamHI/PstI and 3′ PstI/EcoRI fragments into pCIN6 cut withBamHI/EcoRI. This vector contains a T8 signal sequence and 12CA5 HAepitope immediately preceding an in-frame BamHI site.

[0127] Cloning of GABA_(B)-R2, the Novel GABA_(B) Receptor Subtype

[0128] EST clones (H14151, R76089, R80651, AA324303, T07621, Z43654)were identified with approximately 50% nucleotide identity toGABA_(B)-R1. PCR revealed that H14151 contained a 1.5 Kb insert andencoded sufficient sequence for a substantial portion the novel GABA₆receptor. PCR between the 3′ end of H14151 and the 5′ end of AA324303,using a cerebellum cDNA library as template, produced a ˜700 bp product,which when cloned into the T-vector (TA cloning kit, Invitrogen) andsequenced, revealed that T07621 overlaps within M324303. Also, Z43654 aswell as genomic DNA fragments R76089 and R80651 were found to overlapM324303 and together provided sequence data for the 3′ end of the GABA₆subtype receptor. Further sequencing of H14151 provided the fullsequence for the novel receptor subtype. However, because of ambiguitiesin the position of the stop codon in Z43654/R80448/R80651, Incyte clones662098 and 090041, which overlap this region, were sequenced. The stopcodon was identified and sequence for GABA_(B)-R2 was confirmed aswithin H14151 (5′ end) and 662098 (3′ end). 5′ sequences of GABA_(B)-R2were PCR generated using primers 5′-ATGGCTTCCCCGCGGAG-3′ to provide thestart codon of the receptor and primer 5′-GMCAGGCGTGGTIGCAG-3′, primingbeyond a unique EagI site. The expected ˜250 bp product was cloned intopCRSCRIPT and sequenced. Full length receptor was then assembled with athree way ligation between H14151, cut with ApaLI/EagI; 662098, cut withApaLI/NotI and pCRSCRIPT-GABA_(B)-R2-5′ PCR product, restricted byEagI.Full length GABA_(B)-R2 was removed from the pCRSCRIPT vector usingEcoRI/NotI and ligated into pcDNA3 (Invitrogen) for expression studies.

[0129] HA-epitope tagged GABA_(B)-R2 was constructed in pCIN6,.A linkerwas constructed encoding amino acids between the GABA.-R2 signalsequence and the unique EagI site.

[0130] HindIII XhoI EagI EcoRI

[0131] AGCTT CTC GAG GCT TGG GGA TGG GCA CGA GGA GOT CCT GCT CGG CCG G

[0132] A GAG CTC CGA ACC CCT ACC CGT GCT CCT CGT GGT CGA GCC GGC CTT AAAla Trp Gly Trp Ala Arg Gly Ala Pro Arg

[0133] The linker was cloned into pUC18 (EcoRI/HindIII) followed by fulllength GABA_(B)-R2, from pCRSCRIPT as an Eagli/NotI fragment. Finally,the modified GABA_(B)-R2 was cloned into pCIN6 as a XhoI fragment.

[0134] Distribution Studies

[0135] Blots were hybridized overnight at 65° C. according to themanufacturers' instructions with radioactively randomly primed cDNAprobes using ExpressHyb Hybridization solution. Probe for GABA_(B)-R1,corresponding to residues 1129-1618 of the GABA_(B)-R1b coding sequencewas PCR amplified using primers 5′-CGCCTGGAGGACTTCMCTACAA-3′ and5′-TCCTCCCMTGTGGTMCCATCG-3′ against GABA_(B)-R1b DNA as template.GABA_(B)-R2 cDNA probe, corresponding to residues 1397-1800, wasamplified by PCR using primers 5′-ACMGACCATCATCCTGGA-3′ and5′-GATCACMGCAGTTTCTGGTC-3′ with GABA_(B)-R2 DNA as template. DNAfragments were labelled with ³²P-α-dCTP using a Rediprime DNA labellingsystem (Amersham). Probes were labelled to a specific activity of >10⁹cpm/μg and were used at a concentration of approximately 5 ng/mlhybridization solution. Following hybridization, blots were washed with2×SSC/1% SDS at 65° C., and 0.1×SSC/0.5% SDS at 55° C. (20×SSC is 3MNaCl/0.3M Na₃Citrate.2H₂O pH 7.0) and were exposed to X-ray film.

[0136] Yeast Two Hybrid Studies

[0137]Saccharomyces cerevisiae Y190 [MATa, gal4 gal80, ade2-101, his3,trp1-901, ura3-52, leu2-3,112, URA3::GAL1-lacZ, LYS2::GAL1-HIS3,cyh^(R)] was used for all described yeast two hybrid work (Harper etal., 1993, Clontech Laboratories, 1996). GAL4 binding-domain (GAL4BD)fusion vectors were constructed in either pYTH9 (Fuller et al., 1998) orpYTH16, an episomal version of pYTH9. All GAL4 activation-domain fusionswere made in pACT2 (Clontech Laboratories, 1998) All yeast manipulationswere carried out using standard yeast media (Sherman, 1991). Human BrainMATCHMAKER library (HL4004AH) in pACT2 was purchased from ClontechLaboratories and amplified according to the manufacturers' instructions.The GABA_(B)-R1 C-terminal domain was amplified from a full lengthclone, using primers 5′-GTTGTCCCCATGGTGCCCMGATGCGCA GGCTGATCACC-3′ and5′-GTCCTGCGGCCGCGGATCCTCACTTATAAAGCAAATGCACT CG-3′. PCR product wassize-fractionated on 0.8% agarose gel, purified and force-clonedNcoI/NotI into'pYTH9 and subsequently into pACT2. The GABA_(B)-R2C-terminal domain was similarly generated with primers5′-CTCTGCCCCATGGCCGTGCCGAAGCTCATCACCCTGA GAACAAACCC-3′ and5′-GGCCCAGGGCGGCCGCACTTACAGGCCCGAGACCATGACTC GGAAGGAGGG-3′ and subclonedinto pYTH9, pYTH16 and pACT2. All cloned PCR products were sequenced andconfirmed as error free.

[0138] The GAL4_(BD)-GABA_(B)-R1 C-terminus fusion in pYTH9 was stablyintegrated into the trp1 locus of Y190 by targetted homologousrecombination. Yeast expressing GAL4BD-GABA_(B)-R1 C-terminus wereselected and transformed with Human brain cDNA library under leucineselection, using a high efficiency Lithium acetate transformationprotocol (Clontech Laboratories, 1998). Sufficient independent cDNAswere transformed to give a three fold representation of the library.Interacting clones were selected by growth under 20 mM3-amino-1,2,4-triazole (Sigma) selection, followed by production ofβ-galactosidase, as determined by a freeze-fracture assay (ClontechLaboratories, 1998). Plasmid DNA was recovered from yeast cellsfollowing digestion of the cell wall by 400 μg/ml Zymolase 100T (ICNBiochemicals) in 250 μl 1.2M Sorbitol; 0.1 M potassium phosphate buffer(pH 7.4) at 37° for 2 h. Plasmid DNA was extracted by standard Qiagenalkaline lysis miniprep as per manufacturers' instructions andtransformed into Ultracompetent XL-2Blue cells (Stratagene). Plasmid DNAwas sequenced using primer 5′-CAGGGATGTTIMTACCACTACMTGG-3′ usingautomated ABI sequencing and resulting sequences were blasted againstthe databases.

[0139] Yeast Y190 was transformed with pYTH16 and pACT2 expressingGABA_(b)-R1 C-terminal domain and the GABA_(B)-R2 C-terminal domain inall combinations, as well as against empty vectors. Transformants weregrown in liquid media to mid-logarithmic phase and approximately 1.5 mlharvested. β-galactosidase activity was quantified using substrateo-nitrophenyl β-Dgalactopyranoside (ONPG; Sigma) using a liquid nitrogenfreeze fracture regime essentially as described by Harshman et al.,(1988).

[0140] Two-Microelectrode Voltage-Clamp in Xenopus Oocytes

[0141] Adult female Xenopus laevis (Blades Biologicals) wereanaesthetised using 0.2% tricaine (3-aminobenzoic acid ethyl ester),killed and the ovaries rapidly removed. Oocytes were de-folliculated bycollagenase digestion (Sigma type I, 1.5 mg ml⁻¹) in divalentcation-free OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 5 mMHEPES; pH 7.5 at 25° C.). Single stage V and VI oocytes were transferredto ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mMHEPES; pH 7.5 at 25° C.) which contained 50 kg ml⁻¹ gentamycin andstored at 18° C.

[0142] GABA_(B)-R1a, GABA_(B)-R1b (both in pcDNA3.1 rev, Invitrogen),GABA_(B)-R2, GIRK1, GIRK4 (in pcDNA3) and cystic fibrosis transmembraneregulator (CFTR; in pBluescript, Stratagene) were linearised andtranscribed to RNA using T7 or T3 polymerase (Promega Wizard kit).m′G(5′)pp(5′)GTP capped cRNA was injected into oocytes (20-50 nl of 1μgμl⁻¹ RNA per oocyte) and whole-cell currents were recorded usingtwo-microelectrode voltage-clamp (Geneclamp amplifier, Axon instrumentsInc.) 3 to 7 days post-RNA injection. Microelectrodes had a resistanceof 0.5 to 2MΩ when filled with 3M KCl. In all experiments oocytes werevoltage-clamped at a holding potential of −60 mV in ND96 solution(superfused at 2 ml per min.) and agonists were applied by addition tothis extracellular solution. In GIRK experiments the extracellularsolution was changed to a high potassium solution prior to agonistapplication, to facilitate the recording of inward potassium currents.Current-voltage curves were constructed by applying 200 ms voltage-clamppulses from the holding potential of −60 mV to test potentials between−100 mV and +50 mV.

[0143] Mammalian Cell Culture and Transfections

[0144] HEK293T cells (HEK293 cells stably expressing the SV40 largeT-antigen) were maintained in DMEM containing 10% (v/v) foetal calfserum and 2 mM glutamine. Cells were seeded in 60 mm culture dishes andgrown to 60-80% confluency (18-24 h) prior to transfection with pCDNA3containing the relevant DNA species using Lipofectamine reagent. Fortransfection, 3 μg of DNA was mixed with 10 μl of Lipofectamine in 0.2ml of Opti-MEM (Life Technologies Inc.) and was incubated at roomtemperature for 30 min prior to the addition of 1.6 ml of Opti-MEM.Cells were exposed to the Lipofectamine/DNA mixture for 5 h and 2 ml of20% (v/v) newborn calf serum in DMEM was then added. Cells wereharvested 48-72 h after transfection.

[0145] Preparation of Membranes

[0146] Plasma membrane-containing P2 particulate fractions were preparedfrom cell pastes frozen at −80° C. after harvest. All procedures werecarried out at 4° C. Cell pellets were resuspended in 1 ml of 10 mMTris-HCl and 0.1 mM EDTA, pH 7.5 (buffer A) and by homogenisation for 20s with a polytron homogeniser followed by passage (5 times) through a25-guage needle. Cell lysates were centrifuged at 1,000 g for 10 min ina microcentrifuge to pellet the nuclei and unbroken cells and P2particulate fractions were recovered by microcentrifugation at 16,000 gfor 30 min. P2 particulate fractions were resuspended in buffer A andstored at 80° C. until required. Protein concentrations were determinedusing the bicinchoninic acid (BCA) procedure (Smith et al., 1985) usingBSA as a standard.

[0147] High affinity [³⁵S]GTPγS Binding

[0148] Assays were performed in 96-well format using a method modifiedfrom Wieland and Jakobs, 1994. Membranes (10 mg per point) were dilutedto 0.083 mg/ml in assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl₂,pH 7.4) supplemented with saponin (10 mg/l) and pre-incubated with 40 mMGDP. Various concentrations of GABA were added, followed by [³⁵S]GTPgS(1170 Ci/mmol, Amersham) at 0.3 nM (total vol. of 100 ml) and bindingwas allowed to proceed at room temperature for 30 min. Non-specificbinding was determined by the inclusion of 0.6 mM GTP. Wheatgermagglutinin SPA beads (Amersham) (0.5 mg) in 25 ml assay buffer wereadded and the whole was incubated at room temperature for 30 min withagitation. Plates were centrifuged at 1500 g for 5 min and bound[³⁵S]GTPgS was determined by scintillation counting on a Wallac 1450microbeta Trilux scintillation counter.

[0149] Measurement of cAMP Levels

[0150] 24 hours following transfection, each 60 mm dish of HEK293T cellswas split into 36 wells of a 96-well plate and the cells were allowed toreattach overnight. Cells were washed with PBS and pre-incubated in DMEMmedium containing 300 μM IBMX for 30 minutes at 37° C. Forskolin (50 μM)and varying concentrations of GABA were added and cells incubated for afurther 30 min prior to cAMP extraction with 0.1M HCl for 1 h at 4° C.Assays were neutralised with 0.1 M KHCO₃ and cAMP levels determinedusing scintillation proximity assays (Biotrak Kit, Amersham).

[0151] Flow Cytometric Analysis

[0152] HEK293T cells were transiently transfected with cDNA asdescribed. 48-72 h following transfection, cells were recovered andwashed twice in PBS supplemented with 0.1% (w/v) NaN₃ and 2.5% (v/v)foetal calf serum. Cells were resuspended in buffer and incubated withprimary antibodies 9E10 (c-Myc) or 12CA5 (HA) for 15 min at roomtemperature. Following three further washes with PBS, cells wereincubated with secondary antibody (sheep anti-mouse Fab₂ coupled withfluorescein isothiocyanate (FITC)) diluted 1:30 for 15 min at roomtemperature. For permeabilised cells, a Fix and Perm kit (Caltag) wasused. Cell analysis was performed on a Coulter Elite flow-cytometer setup to detect FITC fluoresence. 30,000 cells were analysed for eachsample.

[0153] Immunological Studies

[0154] Antiserum 501 was raised against a synthetic peptidecorresponding to the C-terminal 15 amino acids of the GABA_(B)-R1receptor and-was produced in a sheep, using a conjugate of this peptideand keyhole limpet hemocyanin (Calbiochem) as antigen. Membrane samples30-60 μg) were resolved by SDS-PAGE using 10% (w/v) acrylamide.Following electrophoresis, proteins were subsequently transferred tonitrocellulose (Hybond ECL, Amersham), probed with antiserum 501 at1:1000 dilution and visualised by enhanced chemiluminescence (ECL,Amersham). Epitope tags were visualised by immunoblotting with anti-Myc(9E10; 1:100 dilution) or anti-HA (12CA5; 1:500) monoclonal antibodies.

[0155] Deglycosylation

[0156] Enzymatic removal of asparagine-linked (N-linked) carbohydratemoieties with endoglycosidases F and H was performed essentiallyaccording to manufacturers' instructions (Boehringer Mannheim) using 50μg of membrane protein per enzyme reaction. GABA_(B) receptorglycosylation status was studied following SDS-PAGE/immunoblotting ofsamples.

[0157] Immunoprecipitation Procedures

[0158] Transiently transfected HEK293T cells were harvested as describedabove from 60 mm culture dishes. Cells from each dish were resuspendedin 1 ml of 50 mM Tris-HCl, 150 mM NaCl, 1% (v/v) Nonidet® P40, 0.5%(w/v) sodium deoxycholate, pH 7.5 (lysis buffer) supplemented withCompletes protease inhibitor cocktail tablets (1 tablet/25 ml)(Boehringer Mannheim). Cell lysis and membrane protein solubilisationwas achieved by homogenisation for 20 seconds with a polytronhomogeniser, followed by gentle mixing for 30 min at 4° C. Insolubledebris was removed by microcentrifugation at 16,000 g for 15 min at 4°C. and the supernatant was pre-cleared by incubating with 50 μl ofProtein A-agarose (Boehringer Mannheim) for 3 h at 4° C. on a helicalwheel to reduce non-specific background. Solubilised supematant wasdivided into 2×500 μl aliquots and 20 pi of either HA or Myc antiserawas added to each. Immunoprecipitation was allowed to proceed for 1 h at4° C. on a helical wheel prior to the addition of 50 μl of ProteinA-agarose suspension. Capture of immune complexes was progressedovernight at 4° C. on a helical wheel. Complexes were collected bymicrocentrifugation 12,000 g for 1 min at 4° C. and supematant wasdiscarded. Beads were washed by gentle resuspension and agitationsequentially in 1 ml of 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% (v/v)Nonidete P40 and 0.05% (w/v) sodium deoxycholate followed by 1 ml of 50mM Tris-HCl, pH 7.5, 0.1% (v/v) Nonidet® P40 and 0.05% (w/v) sodiumdeoxycholate. Immunoprecipitated proteins were released from ProteinA-agarose by incubation in 30 μl of SDS-PAGE sample buffer at 70° C. for10 min and analysed by SDS-PAGE followed by immunoblotting.

[0159] Binding Assays

[0160] Competition binding assays were performed in 50 mM Tris HClbuffer (pH 7.4) containing 40 μM isoguvacine (Tocris Cookson) to blockrat brain GABA_(B) binding sites. P2 membrane preparations were madefrom HEK293T cells transfected using conditions described above.Increasing concentrations of GABA were added to displace the antagonist[3H]-CGP 54626 (Tocris Cookson, 40Ci/mmol). Assay conditions were0.4-0.6 nM [³H]-CGP54626, incubated with 50 μg/tube crude rat brain‘mitochondrial’ fractions or 25 μg/tube HEK293T P2 membranes atroom-temperature for 20 minutes. The total volume per tube was 0.5 mland non specific binding was determined using 1 mM GABA. Bound ligandwas recovered using a Brandel 48 well harvester onto GF/B filters(Whatman) and measured by liquid scintillation using a Beckman LS6500counter.

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1 55 1 26 DNA Artificial Sequence primer 1 gcgactgctg tgggctgctt actggc26 2 30 DNA Artificial Sequence primer 2 gcgaattccc tgtcctccctcaccctaccc 30 3 24 DNA Artificial Sequence primer 3 ccgagctcaagctcatccac cacg 24 4 24 DNA Artificial Sequence primer 4 tcttcctccactccttcttt tctt 24 5 27 DNA Artificial Sequence primer 5 tgagctggagccataggaaa gcacaat 27 6 26 DNA Artificial Sequence primer 6 gatcttgatagggtcgttgt agagca 26 7 20 DNA Artificial Sequence primer 7 gctcctaacgctccccaaca 20 8 20 DNA Artificial Sequence primer 8 ggcctggatcacacttgctg 20 9 19 DNA Artificial Sequence primer 9 cccaacgcca cctcagaag19 10 19 DNA Artificial Sequence primer 10 ccgctcatgg gaaacagtg 19 11 27DNA Artificial Sequence primer 11 ccccggcaca catactcaat ctcatag 27 12 22DNA Artificial Sequence primer 12 ccggtacctg atgccccctt cc 22 13 22 DNAArtificial Sequence primer 13 cgagatgttg ctgctgctgc ta 22 14 23 DNAArtificial Sequence primer 14 taggatccca ctccccccat ccc 23 15 20 DNAArtificial Sequence primer 15 ccagcgtgga gacagagctg 20 16 17 DNAArtificial Sequence primer 16 atggcttccc cgcggag 17 17 18 DNA ArtificialSequence primer 17 gaacaggcgt ggttgcag 18 18 48 DNA Artificial Sequenceconstructed oligo 18 agcttctcga ggcttgggga tgggcacgag gagctcctgctcggccgg 48 19 48 DNA Artificial Sequence constructed oligo 19agagctccga acccctaccc gtgctcctcg tggtcgagcc ggccttaa 48 20 10 PRTArtificial Sequence synthetic peptide 20 Ala Trp Gly Trp Ala Arg Gly AlaPro Arg 1 5 10 21 23 DNA Artificial Sequence primer 21 cgcctggaggacttcaacta caa 23 22 23 DNA Artificial Sequence primer 22 tcctcccaatgtggtaacca tcg 23 23 19 DNA Artificial Sequence primer 23 acaagaccatcatcctgga 19 24 21 DNA Artificial Sequence primer 24 gatcacaagcagtttctggt c 21 25 39 DNA Artificial Sequence primer 25 gttgtccccatggtgcccaa gatgcgcagg ctgatcacc 39 26 43 DNA Artificial Sequence primer26 gtcctgcggc cgcggatcct cacttataaa gcaaatgcac tcg 43 27 47 DNAArtificial Sequence primer 27 ctctgcccca tggccgtgcc gaagctcatcaccctgagaa caaaccc 47 28 51 DNA Artificial Sequence primer 28 ggcccagggcggccgcactt acaggcccga gaccatgact cggaaggagg g 51 29 27 DNA ArtificialSequence primer 29 cagggatgtt taataccact acaatgg 27 30 2826 DNA Homosapiens 30 atggcttccc cgcggagctc cgggcagccc gggccgccgc cgccgccgccaccgccgccc 60 gcgcgcctgc tactgctact gctgctgccg ctgctgctgc ctctggcgcccggggcctgg 120 ggctgggcgc ggggcgcccc ccggccgccg cccagcagcc cgccgctctccatcatgggc 180 ctcatgccgc tcaccaagga ggtggccaag ggcagcatcg ggcgcggtgtgctccccgcc 240 gtggaactgg ccatcgagca gatccgcaac gagtcactcc tgcgcccctacttcctcgac 300 ctgcggctct atgacacgga gtgcgacaac gcaaaagggt tgaaagccttctacgatgca 360 ataaaatacg ggcctaacca cttgatggtg tttggaggcg tctgtccatccgtcacatcc 420 atcattgcag agtccctcca aggctggaat ctggtgcagc tttcttttgctgcaaccacg 480 cctgttctag ccgataagaa aaaataccct tatttctttc ggaccgtcccatcagacaat 540 gcggtgaatc cagccattct gaagttgctc aagcactacc agtggaagcgcgtgggcacg 600 ctgacgcaag acgttcagag gttctctgag gtgcggaatg acctgactggagttctgtat 660 ggcgaggaca ttgagatttc agacaccgag agcttctcca acgatccctgtaccagtgtc 720 aaaaagctga aggggaatga tgtgcggatc atccttggcc agtttgaccagaatatggca 780 gcaaaagtgt tctgttgtgc atacgaggag aacatgtatg gtagtaaatatcagtggatc 840 attccgggct ggtacgagcc ttcttggtgg gagcaggtgc acacggaagccaactcatcc 900 cgctgcctcc ggaagaatct gcttgctgcc atggagggct acattggcgtggatttcgag 960 cccctgagct ccaagcagat caagaccatc tcaggaaaga ctccacagcagtatgagaga 1020 gagtacaaca acaagcggtc aggcgtgggg cccagcaagt tccacgggtacgcctacgat 1080 ggcatctggg tcatcgccaa gacactgcag agggccatgg agacactgcatgccagcagc 1140 cggcaccagc ggatccagga cttcaactac acggaccaca cgctgggcaggatcatcctc 1200 aatgccatga acgagaccaa cttcttcggg gtcacgggtc aagttgtattccggaatggg 1260 gagagaatgg ggaccattaa atttactcaa tttcaagaca gcagggaggtgaaggtggga 1320 gagtacaacg ctgtggccga cacactggag atcatcaatg acaccatcaggttccaagga 1380 tccgaaccac caaaagacaa gaccatcatc ctggagcagc tgcggaagatctccctacct 1440 ctctacagca tcctctctgc cctcaccatc ctcgggatga tcatggccagtgcttttctc 1500 ttcttcaaca tcaagaaccg gaatcagaag ctcataaaga tgtcgagtccatacatgaac 1560 aaccttatca tccttggagg gatgctctcc tatgcttcca tatttctctttggccttgat 1620 ggatcctttg tctctgaaaa gacctttgaa acactttgca ccgtcaggacctggattctc 1680 accgtgggct acacgaccgc ttttggggcc atgtttgcaa agacctggagagtccacgcc 1740 atcttcaaaa atgtgaaaat gaagaagaag atcatcaagg accagaaactgcttgtgatc 1800 gtggggggca tgctgctgat cgacctgtgt atcctgatct gctggcaggctgtggacccc 1860 ctgcgaagga cagtggagaa gtacagcatg gagccggacc cagcaggacgggatatctcc 1920 atccgccctc tcctggagca ctgtgagaac acccatatga ccatctggcttggcatcgtc 1980 tatgcctaca agggacttct catgttgttc ggttgtttct tagcttgggagacccgcaac 2040 gtcagcatcc ccgcactcaa cgacagcaag tacatcggga tgagtgtctacaacgtgggg 2100 atcatgtgca tcatcggggc cgctgtctcc ttcctgaccc gggaccagcccaatgtgcag 2160 ttctgcatcg tggctctggt catcatcttc tgcagcacca tcaccctctgcctggtattc 2220 gtgccgaagc tcatcaccct gagaacaaac ccagatgcag caacgcagaacaggcgattc 2280 cagttcactc agaatcagaa gaaagaagat tctaaaacgt ccacctcggtcaccagtgtg 2340 aaccaagcca gcacatcccg cctggagggc ctacagtcag aaaaccatcgcctgcgaatg 2400 aagatcacag agctggataa agacttggaa gaggtcacca tgcagctgcaggacacacca 2460 gaaaagacca cctacattaa acagaaccac taccaagagc tcaatgacatcctcaacctg 2520 ggaaacttca ctgagagcac agatggagga aaggccattt taaaaaatcacctcgatcaa 2580 aatccccagc tacagtggaa cacaacagag ccctctcgaa catgcaaagatcctatagaa 2640 gatataaact ctccagaaca catccagcgt cggctgtccc tccagctccccatcctccac 2700 cacgcctacc tcccatccat cggaggcgtg gacgccagct gtgtcagcccctgcgtcagc 2760 cccaccgcca gcccccgcca cagacatgtg ccaccctcct tccgagtcatggtctcgggc 2820 ctgtaa 2826 31 941 PRT Homo sapiens 31 Met Ala Ser ProArg Ser Ser Gly Gln Pro Gly Pro Pro Pro Pro Pro 1 5 10 15 Pro Pro ProPro Ala Arg Leu Leu Leu Leu Leu Leu Leu Pro Leu Leu 20 25 30 Leu Pro LeuAla Pro Gly Ala Trp Gly Trp Ala Arg Gly Ala Pro Arg 35 40 45 Pro Pro ProSer Ser Pro Pro Leu Ser Ile Met Gly Leu Met Pro Leu 50 55 60 Thr Lys GluVal Ala Lys Gly Ser Ile Gly Arg Gly Val Leu Pro Ala 65 70 75 80 Val GluLeu Ala Ile Glu Gln Ile Arg Asn Glu Ser Leu Leu Arg Pro 85 90 95 Tyr PheLeu Asp Leu Arg Leu Tyr Asp Thr Glu Cys Asp Asn Ala Lys 100 105 110 GlyLeu Lys Ala Phe Tyr Asp Ala Ile Lys Tyr Gly Pro Asn His Leu 115 120 125Met Val Phe Gly Gly Val Cys Pro Ser Val Thr Ser Ile Ile Ala Glu 130 135140 Ser Leu Gln Gly Trp Asn Leu Val Gln Leu Ser Phe Ala Ala Thr Thr 145150 155 160 Pro Val Leu Ala Asp Lys Lys Lys Tyr Pro Tyr Phe Phe Arg ThrVal 165 170 175 Pro Ser Asp Asn Ala Val Asn Pro Ala Ile Leu Lys Leu LeuLys His 180 185 190 Tyr Gln Trp Lys Arg Val Gly Thr Leu Thr Gln Asp ValGln Arg Phe 195 200 205 Ser Glu Val Arg Asn Asp Leu Thr Gly Val Leu TyrGly Glu Asp Ile 210 215 220 Glu Ile Ser Asp Thr Glu Ser Phe Ser Asn AspPro Cys Thr Ser Val 225 230 235 240 Lys Lys Leu Lys Gly Asn Asp Val ArgIle Ile Leu Gly Gln Phe Asp 245 250 255 Gln Asn Met Ala Ala Lys Val PheCys Cys Ala Tyr Glu Glu Asn Met 260 265 270 Tyr Gly Ser Lys Tyr Gln TrpIle Ile Pro Gly Trp Tyr Glu Pro Ser 275 280 285 Trp Trp Glu Gln Val HisThr Glu Ala Asn Ser Ser Arg Cys Leu Arg 290 295 300 Lys Asn Leu Leu AlaAla Met Glu Gly Tyr Ile Gly Val Asp Phe Glu 305 310 315 320 Pro Leu SerSer Lys Gln Ile Lys Thr Ile Ser Gly Lys Thr Pro Gln 325 330 335 Gln TyrGlu Arg Glu Tyr Asn Asn Lys Arg Ser Gly Val Gly Pro Ser 340 345 350 LysPhe His Gly Tyr Ala Tyr Asp Gly Ile Trp Val Ile Ala Lys Thr 355 360 365Leu Gln Arg Ala Met Glu Thr Leu His Ala Ser Ser Arg His Gln Arg 370 375380 Ile Gln Asp Phe Asn Tyr Thr Asp His Thr Leu Gly Arg Ile Ile Leu 385390 395 400 Asn Ala Met Asn Glu Thr Asn Phe Phe Gly Val Thr Gly Gln ValVal 405 410 415 Phe Arg Asn Gly Glu Arg Met Gly Thr Ile Lys Phe Thr GlnPhe Gln 420 425 430 Asp Ser Arg Glu Val Lys Val Gly Glu Tyr Asn Ala ValAla Asp Thr 435 440 445 Leu Glu Ile Ile Asn Asp Thr Ile Arg Phe Gln GlySer Glu Pro Pro 450 455 460 Lys Asp Lys Thr Ile Ile Leu Glu Gln Leu ArgLys Ile Ser Leu Pro 465 470 475 480 Leu Tyr Ser Ile Leu Ser Ala Leu ThrIle Leu Gly Met Ile Met Ala 485 490 495 Ser Ala Phe Leu Phe Phe Asn IleLys Asn Arg Asn Gln Lys Leu Ile 500 505 510 Lys Met Ser Ser Pro Tyr MetAsn Asn Leu Ile Ile Leu Gly Gly Met 515 520 525 Leu Ser Tyr Ala Ser IlePhe Leu Phe Gly Leu Asp Gly Ser Phe Val 530 535 540 Ser Glu Lys Thr PheGlu Thr Leu Cys Thr Val Arg Thr Trp Ile Leu 545 550 555 560 Thr Val GlyTyr Thr Thr Ala Phe Gly Ala Met Phe Ala Lys Thr Trp 565 570 575 Arg ValHis Ala Ile Phe Lys Asn Val Lys Met Lys Lys Lys Ile Ile 580 585 590 LysAsp Gln Lys Leu Leu Val Ile Val Gly Gly Met Leu Leu Ile Asp 595 600 605Leu Cys Ile Leu Ile Cys Trp Gln Ala Val Asp Pro Leu Arg Arg Thr 610 615620 Val Glu Lys Tyr Ser Met Glu Pro Asp Pro Ala Gly Arg Asp Ile Ser 625630 635 640 Ile Arg Pro Leu Leu Glu His Cys Glu Asn Thr His Met Thr IleTrp 645 650 655 Leu Gly Ile Val Tyr Ala Tyr Lys Gly Leu Leu Met Leu PheGly Cys 660 665 670 Phe Leu Ala Trp Glu Thr Arg Asn Val Ser Ile Pro AlaLeu Asn Asp 675 680 685 Ser Lys Tyr Ile Gly Met Ser Val Tyr Asn Val GlyIle Met Cys Ile 690 695 700 Ile Gly Ala Ala Val Ser Phe Leu Thr Arg AspGln Pro Asn Val Gln 705 710 715 720 Phe Cys Ile Val Ala Leu Val Ile IlePhe Cys Ser Thr Ile Thr Leu 725 730 735 Cys Leu Val Phe Val Pro Lys LeuIle Thr Leu Arg Thr Asn Pro Asp 740 745 750 Ala Ala Thr Gln Asn Arg ArgPhe Gln Phe Thr Gln Asn Gln Lys Lys 755 760 765 Glu Asp Ser Lys Thr SerThr Ser Val Thr Ser Val Asn Gln Ala Ser 770 775 780 Thr Ser Arg Leu GluGly Leu Gln Ser Glu Asn His Arg Leu Arg Met 785 790 795 800 Lys Ile ThrGlu Leu Asp Lys Asp Leu Glu Glu Val Thr Met Gln Leu 805 810 815 Gln AspThr Pro Glu Lys Thr Thr Tyr Ile Lys Gln Asn His Tyr Gln 820 825 830 GluLeu Asn Asp Ile Leu Asn Leu Gly Asn Phe Thr Glu Ser Thr Asp 835 840 845Gly Gly Lys Ala Ile Leu Lys Asn His Leu Asp Gln Asn Pro Gln Leu 850 855860 Gln Trp Asn Thr Thr Glu Pro Ser Arg Thr Cys Lys Asp Pro Ile Glu 865870 875 880 Asp Ile Asn Ser Pro Glu His Ile Gln Arg Arg Leu Ser Leu GlnLeu 885 890 895 Pro Ile Leu His His Ala Tyr Leu Pro Ser Ile Gly Gly ValAsp Ala 900 905 910 Ser Cys Val Ser Pro Cys Val Ser Pro Thr Ala Ser ProArg His Arg 915 920 925 His Val Pro Pro Ser Phe Arg Val Met Val Ser GlyLeu 930 935 940 32 72 PRT Homo sapiens 32 Met Leu Leu Leu Leu Leu AlaPro Leu Phe Leu Arg Pro Pro Gly Ala 1 5 10 15 Gly Gly Ala Gln Thr ProAsn Ala Thr Ser Glu Gly Cys Gln Ile Ile 20 25 30 His Pro Pro Trp Glu GlyGly Ile Arg Tyr Arg Gly Leu Thr Arg Asp 35 40 45 Gln Val Lys Ala Ile AsnPhe Leu Pro Val Asp Tyr Glu Ile Glu Tyr 50 55 60 Val Cys Arg Gly Glu ArgGlu Val 65 70 33 29 PRT Homo sapiens 33 Met Gly Pro Gly Ala Pro Phe AlaArg Val Gly Trp Pro Leu Pro Leu 1 5 10 15 Leu Val Val Met Ala Ala GlyVal Ala Pro Val Trp Ala 20 25 34 38 PRT Homo sapiens 34 Met Ala Ser ProArg Ser Ser Gly Gln Pro Gly Pro Pro Pro Pro Pro 1 5 10 15 Pro Pro ProPro Ala Arg Leu Leu Leu Leu Leu Leu Leu Pro Leu Leu 20 25 30 Leu Pro LeuAla Pro Gly 35 35 91 PRT Homo sapiens 35 Val Gly Pro Lys Val Arg Lys CysLeu Ala Asn Gly Ser Trp Thr Asp 1 5 10 15 Met Asp Thr Pro Ser Arg CysVal Arg Ile Cys Ser Lys Ser Tyr Leu 20 25 30 Thr Leu Glu Asn Gly Lys ValPhe Leu Thr Gly Gly Asp Leu Pro Ala 35 40 45 Leu Asp Gly Ala Arg Val AspPhe Arg Cys Asp Pro Asp Phe His Leu 50 55 60 Val Gly Ser Ser Arg Ser IleCys Ser Gln Gly Gln Trp Ser Thr Pro 65 70 75 80 Lys Pro His Cys Gln ValAsn Arg Thr Pro His 85 90 36 9 PRT Homo sapiens 36 Ser Glu Arg Arg AlaVal Tyr Ile Gly 1 5 37 18 PRT Homo sapien 37 Ser His Ser Pro His Leu ProArg Pro His Ser Arg Val Pro Pro His 1 5 10 15 Pro Ser 38 24 PRT Homosapien 38 Ala Trp Gly Trp Ala Arg Gly Ala Pro Arg Pro Pro Pro Ser SerPro 1 5 10 15 Pro Leu Ser Ile Met Gly Leu Met 20 39 100 PRT Homo sapiens39 Ala Leu Phe Pro Met Ser Gly Gly Trp Pro Gly Gly Gln Ala Cys Gln 1 510 15 Pro Ala Val Glu Met Ala Leu Glu Asp Val Asn Ser Arg Arg Asp Ile 2025 30 Leu Pro Asp Tyr Glu Leu Lys Leu Ile His His Asp Ser Lys Cys Asp 3540 45 Pro Gly Gln Ala Thr Lys Tyr Leu Tyr Glu Leu Leu Tyr Asn Asp Pro 5055 60 Ile Lys Ile Ile Leu Met Pro Gly Cys Ser Ser Val Ser Thr Leu Val 6570 75 80 Ala Glu Ala Ala Arg Met Trp Asn Leu Ile Val Leu Ser Tyr Gly Ser85 90 95 Ser Ser Pro Ala 100 40 100 PRT Homo sapiens 40 Pro Leu Thr LysGlu Val Ala Lys Gly Ser Ile Gly Arg Gly Val Leu 1 5 10 15 Pro Ala ValGlu Leu Ala Ile Glu Gln Ile Arg Asn Glu Ser Leu Leu 20 25 30 Arg Pro TyrPhe Leu Asp Leu Arg Leu Tyr Asp Thr Glu Cys Asp Asn 35 40 45 Ala Lys GlyLeu Lys Ala Phe Tyr Asp Ala Ile Lys Tyr Gly Pro Asn 50 55 60 His Leu MetVal Phe Gly Gly Val Cys Pro Ser Val Thr Ser Ile Ile 65 70 75 80 Ala GluSer Leu Gln Gly Trp Asn Leu Val Gln Leu Ser Phe Ala Ala 85 90 95 Thr ThrPro Val 100 41 101 PRT Homo sapien 41 Leu Ser Asn Arg Gln Arg Phe ProThr Phe Phe Arg Thr His Pro Ser 1 5 10 15 Ala Thr Leu His Asn Pro ThrArg Val Lys Leu Phe Glu Lys Trp Gly 20 25 30 Trp Lys Lys Ile Ala Thr IleGln Gln Thr Thr Glu Val Phe Thr Ser 35 40 45 Thr Leu Asp Asp Leu Glu GluArg Val Lys Glu Ala Gly Ile Glu Ile 50 55 60 Thr Phe Arg Gln Ser Phe PheSer Asp Pro Ala Val Pro Val Lys Asn 65 70 75 80 Leu Lys Arg Gln Asp AlaArg Ile Ile Val Gly Leu Phe Tyr Glu Thr 85 90 95 Glu Ala Arg Lys Val 10042 101 PRT Homo Sapien 42 Leu Ala Asp Lys Lys Lys Tyr Pro Tyr Phe PheArg Thr Val Pro Ser 1 5 10 15 Asp Asn Ala Val Asn Pro Ala Ile Leu LysLeu Leu Lys His Tyr Gln 20 25 30 Trp Lys Arg Val Gly Thr Leu Thr Gln AspVal Gln Arg Phe Ser Glu 35 40 45 Val Arg Asn Asp Leu Thr Gly Val Leu TyrGly Glu Asp Ile Glu Ile 50 55 60 Ser Asp Thr Glu Ser Phe Ser Asn Asp ProCys Thr Ser Val Lys Lys 65 70 75 80 Leu Lys Gly Asn Asp Val Arg Ile IleLeu Gly Gln Phe Asp Gln Asn 85 90 95 Met Ala Ala Lys Val 100 43 97 PRTHomo sapien 43 Phe Cys Glu Val Tyr Lys Glu Arg Leu Phe Gly Lys Lys TyrVal Trp 1 5 10 15 Phe Leu Ile Gly Trp Tyr Ala Asp Asn Trp Phe Lys IleTyr Asp Pro 20 25 30 Ser Ile Asn Cys Thr Val Asp Glu Met Thr Glu Ala ValGlu Gly His 35 40 45 Ile Thr Thr Glu Ile Val Met Leu Asn Pro Ala Asn ThrArg Ser Ile 50 55 60 Ser Asn Met Thr Ser Gln Glu Phe Val Glu Lys Leu ThrLys Arg Leu 65 70 75 80 Lys Arg His Pro Glu Glu Thr Gly Gly Phe Gln GluAla Pro Leu Ala 85 90 95 Tyr 44 96 PRT Homo sapien 44 Phe Cys Cys AlaTyr Glu Glu Asn Met Tyr Gly Ser Lys Tyr Gln Trp 1 5 10 15 Ile Ile ProGly Trp Tyr Glu Pro Ser Trp Trp Glu Gln Val His Thr 20 25 30 Glu Ala AsnSer Ser Arg Cys Leu Arg Lys Asn Leu Leu Ala Ala Met 35 40 45 Glu Gly TyrIle Gly Val Asp Phe Glu Pro Leu Ser Ser Lys Gln Ile 50 55 60 Lys Thr IleSer Gly Lys Thr Pro Gln Gln Tyr Glu Arg Glu Tyr Asn 65 70 75 80 Asn LysArg Ser Gly Val Gly Pro Ser Lys Phe His Gly Tyr Ala Tyr 85 90 95 45 99PRT Homo sapien 45 Asp Ala Ile Trp Ala Leu Ala Leu Ala Leu Asn Lys ThrSer Gly Gly 1 5 10 15 Gly Gly Arg Ser Gly Val Arg Leu Glu Asp Phe AsnTyr Asn Asn Gln 20 25 30 Thr Ile Thr Asp Gln Ile Tyr Arg Ala Met Asn SerSer Ser Phe Glu 35 40 45 Gly Val Ser Gly His Val Val Phe Asp Ala Ser GlySer Arg Met Ala 50 55 60 Trp Thr Leu Ile Glu Gln Pro Gln Gly Gly Ser TyrLys Lys Ile Gly 65 70 75 80 Tyr Tyr Asp Ser Thr Lys Asp Asp Leu Ser TrpSer Lys Thr Asp Lys 85 90 95 Trp Ile Gly 46 100 PRT Homo sapien 46 AspGly Ile Trp Val Ile Ala Lys Thr Leu Gln Arg Ala Met Glu Thr 1 5 10 15Leu His Ala Ser Ser Arg His Gln Arg Ile Gln Asp Phe Asn Tyr Thr 20 25 30Asp His Thr Leu Gly Arg Ile Ile Leu Asn Ala Met Asn Glu Thr Asn 35 40 45Phe Phe Gly Val Thr Gly Gln Val Val Phe Arg Asn Gly Glu Arg Met 50 55 60Gly Thr Ile Lys Phe Thr Gln Phe Gln Asp Ser Arg Glu Val Lys Val 65 70 7580 Gly Glu Tyr Asn Ala Val Ala Asp Thr Leu Glu Ile Ile Asn Asp Thr 85 9095 Ile Arg Phe Gln 100 47 101 PRT Homo sapien 47 Gly Ser Pro Pro Ala AspGln Thr Leu Val Ile Lys Thr Phe Arg Phe 1 5 10 15 Leu Ser Gln Lys LeuPhe Ile Ser Val Ser Val Leu Ser Ser Leu Gly 20 25 30 Ile Val Leu Ala ValVal Cys Leu Ser Phe Asn Ile Tyr Asn Ser His 35 40 45 Val Arg Tyr Ile GlnAsn Ser Gln Pro Asn Leu Asn Asn Leu Thr Ala 50 55 60 Val Gly Cys Ser LeuAla Leu Ala Ala Val Phe Pro Leu Gly Leu Asp 65 70 75 80 Gly Tyr His IleGly Arg Asn Gln Phe Pro Phe Val Cys Gln Ala Arg 85 90 95 Leu Trp Leu LeuGly 100 48 102 PRT Homo sapien 48 Gly Ser Glu Pro Pro Lys Asp Lys ThrIle Ile Leu Glu Gln Leu Arg 1 5 10 15 Lys Ile Ser Leu Pro Leu Tyr SerIle Leu Ser Ala Leu Thr Ile Leu 20 25 30 Gly Met Ile Met Ala Ser Ala PheLeu Phe Phe Asn Ile Lys Asn Arg 35 40 45 Asn Gln Lys Leu Ile Lys Met SerSer Pro Tyr Met Asn Asn Leu Ile 50 55 60 Ile Leu Gly Gly Met Leu Ser TyrAla Ser Ile Phe Leu Phe Gly Leu 65 70 75 80 Asp Gly Ser Phe Val Ser GluLys Thr Phe Glu Thr Leu Cys Thr Val 85 90 95 Arg Thr Trp Ile Leu Thr 10049 102 PRT Homo sapien 49 Leu Gly Phe Ser Leu Gly Tyr Gly Ser Met PheThr Lys Ile Trp Trp 1 5 10 15 Val His Thr Gly Phe Thr Lys Lys Glu GluLys Lys Glu Trp Arg Lys 20 25 30 Thr Leu Glu Pro Trp Lys Leu Tyr Ala ThrVal Gly Leu Leu Val Gly 35 40 45 Met Asp Val Leu Thr Leu Ala Ile Trp GlnIle Val Asp Pro Leu His 50 55 60 Arg Thr Ile Glu Thr Phe Ala Lys Glu GluPro Lys Glu Asp Ile Asp 65 70 75 80 Val Ser Ile Leu Pro Gln Leu Glu HisCys Ser Ser Arg Lys Met Asn 85 90 95 Thr Trp Leu Gly Ile Phe 100 50 99PRT Homo sapien 50 Val Gly Tyr Thr Thr Ala Phe Gly Ala Met Phe Ala LysThr Trp Arg 1 5 10 15 Val His Ala Ile Phe Lys Asn Val Lys Met Lys LysLys Ile Ile Lys 20 25 30 Asp Gln Lys Leu Leu Val Ile Val Gly Gly Met LeuLeu Ile Asp Leu 35 40 45 Cys Ile Leu Ile Cys Trp Gln Ala Val Asp Pro LeuArg Arg Thr Val 50 55 60 Glu Lys Tyr Ser Met Glu Pro Asp Pro Ala Gly ArgAsp Ile Ser Ile 65 70 75 80 Arg Pro Leu Leu Glu His Cys Glu Asn Thr HisMet Thr Ile Trp Leu 85 90 95 Gly Ile Val 51 92 PRT Homo sapien 51 TyrGly Tyr Lys Gly Leu Leu Leu Leu Leu Gly Ile Phe Leu Ala Tyr 1 5 10 15Glu Thr Lys Ser Val Ser Thr Glu Lys Ile Asn Asp His Arg Ala Val 20 25 30Gly Met Ala Ile Tyr Asn Val Ala Val Leu Cys Leu Ile Thr Ala Pro 35 40 45Val Thr Met Ile Leu Ser Ser Gln Gln Asp Ala Ala Phe Ala Phe Ala 50 55 60Ser Leu Ala Ile Val Phe Ser Ser Tyr Ile Thr Leu Val Val Leu Phe 65 70 7580 Val Pro Lys Met Arg Arg Leu Ile Thr Arg Gly Glu 85 90 52 99 PRT Homosapien 52 Tyr Ala Tyr Lys Gly Leu Leu Met Leu Phe Gly Cys Phe Leu AlaTrp 1 5 10 15 Glu Thr Arg Asn Val Ser Ile Pro Ala Leu Asn Asp Ser LysTyr Ile 20 25 30 Gly Met Ser Val Tyr Asn Val Gly Ile Met Cys Ile Ile GlyAla Ala 35 40 45 Val Ser Phe Leu Thr Arg Asp Gln Pro Asn Val Gln Phe CysIle Val 50 55 60 Ala Leu Val Ile Ile Phe Cys Ser Thr Ile Thr Leu Cys LeuVal Phe 65 70 75 80 Val Pro Lys Leu Ile Thr Leu Arg Thr Asn Pro Asp AlaAla Thr Gln 85 90 95 Asn Arg Arg 53 96 PRT Homo sapien 53 Trp Gln SerGlu Ala Gln Asp Thr Met Lys Thr Gly Ser Ser Thr Asn 1 5 10 15 Asn AsnGlu Glu Glu Lys Ser Arg Leu Leu Glu Lys Glu Asn Arg Glu 20 25 30 Leu GluLys Ile Ile Ala Glu Lys Glu Glu Arg Val Ser Glu Leu Arg 35 40 45 His GlnLeu Gln Ser Arg Gln Gln Leu Arg Ser Arg Arg His Pro Pro 50 55 60 Thr ProPro Glu Pro Ser Gly Gly Leu Pro Arg Gly Pro Pro Glu Pro 65 70 75 80 ProAsp Arg Leu Ser Cys Asp Gly Ser Arg Val His Leu Leu Tyr Lys 85 90 95 54102 PRT Homo sapien 54 Phe Gln Phe Thr Gln Asn Gln Lys Lys Glu Asp SerLys Thr Ser Thr 1 5 10 15 Ser Val Thr Ser Val Asn Gln Ala Ser Thr SerArg Leu Glu Gly Leu 20 25 30 Gln Ser Glu Asn His Arg Leu Arg Met Lys IleThr Glu Leu Asp Lys 35 40 45 Asp Leu Glu Glu Val Thr Met Gln Leu Gln AspThr Pro Glu Lys Thr 50 55 60 Thr Tyr Ile Lys Gln Asn His Tyr Gln Glu LeuAsn Asp Ile Leu Asn 65 70 75 80 Leu Gly Asn Phe Thr Glu Ser Thr Asp GlyGly Lys Ala Ile Leu Lys 85 90 95 Asn His Leu Asp Gln Asn 100 55 80 PRTHomo sapien 55 Pro Gln Leu Gln Trp Asn Thr Thr Glu Pro Ser Arg Thr CysLys Asp 1 5 10 15 Pro Ile Glu Asp Ile Asn Ser Pro Glu His Ile Gln ArgArg Leu Ser 20 25 30 Leu Gln Leu Pro Ile Leu His His Ala Tyr Leu Pro SerIle Gly Gly 35 40 45 Val Asp Ala Ser Cys Val Ser Pro Cys Val Ser Pro ThrAla Ser Pro 50 55 60 Arg His Arg His Val Pro Pro Ser Phe Arg Val Met ValSer Gly Leu 65 70 75 80

1. An isolated GABA_(B)-R2 receptor protein or a variant thereof.
 2. Anisolated GABA_(B)-R2 receptor protein having amino acid sequenceprovided in FIG. 1B, or a variant thereof.
 3. A nucleotide sequenceencoding a GABA_(B)-R2 receptor or a variant thereof, or a nucleotidesequence which is complementary thereto.
 4. The nucleotide sequence ofclaim 3 which is a cDNA sequence.
 5. A nucleotide sequence encoding aGABA_(B)-R2 receptor, as shown in FIG. 1A, or a variant thereof, or anucleotide sequence which is complementary thereto.
 6. The nucleotidesequence of claim 4 which is a cDNA sequence.
 7. An expression vectorcomprising the nucleotide sequence of claim 3, which is capable ofexpressing a GABA_(B)-R2 receptor protein or a variant thereof.
 8. Anexpression vector comprising the nucleotide sequence of claim 5, whichis capable of expressing a GABA_(B)-R2 receptor protein or a variantthereof.
 9. A stable cell line comprising a vector according to claim 7.10. The cell line according to claim 9 which is a modified HEK293T cellline.
 11. An antibody specific for a protein as claimed in claim
 1. 12.An antibody specific for a protein as claimed in claim
 2. 13. Anisolated GABA_(B)-R1c receptor protein or a variant thereof.
 14. Anisolated GABA_(B)-R1c receptor protein having amino acid sequenceprovided in FIG. 2, or a variant thereof.
 15. A nucleotide sequenceencoding a GABA_(B)-R1c receptor protein or a variant thereof, or anucleotide sequence which is complementary thereto.
 16. A nucleotidesequence of claim 15 which is a cDNA sequence.
 17. A nucleotide sequenceencoding a GABA_(B)-R1c receptor protein or a variant thereof as claimedin claim 14, or a nucleotide sequence which is complementary thereto.18. A nucleotide sequence of claim 17 which is a cDNA sequence.
 19. Anexpression vector comprising the nucleotide sequence of claim 15, whichis capable of expressing a GABA_(B)-R1c receptor protein or a variantthereof.
 20. A stable cell line comprising the vector according to claim19.
 21. The cell line according to claim 20 which is a modified HEK293Tcell line.
 22. An antibody specific for the protein claimed in claim 13.23. An antibody specific for the protein claimed in claim
 14. 24. AGABA_(B) receptor comprising an heterodimer between a GABA_(B)-R1receptor protein or a variant thereof and a GABA_(B)-R2 receptor proteinor a variant thereof.
 25. The GABA_(B) receptor according to claim 24wherein the GABA_(B)-R1 receptor is a GABA_(B)-R1a receptor or variantthereof.
 26. The GABA_(B) receptor according to claim 24 wherein theGABA_(B)-R1 receptor is a GABA_(B)-R1b receptor or variant thereof. 27.The GABA_(B) receptor according to claim 24 wherein the GABA_(B)-R1receptor is a GABA_(B)-R1c receptor or variant thereof.
 28. Anexpression vector comprising a nucleotide sequence encoding for aGABA_(B)-R1 receptor or a variant thereof and a nucleotide sequenceencoding for a GABA_(B)-R2 receptor or variant thereof, said vectorbeing capable of expressing both a GABA_(B)-R1 and a GABA_(B)-R2receptor proteins or variants thereof.
 29. The vector according to claim28 wherein the GABA_(B)-R1 receptor is a GABA_(B)-R1a receptor orvariant thereof.
 30. The vector according to claim 28 wherein theGABA_(B)-R1 receptor is a GABA_(B)-R1b receptor or variant thereof. 31.The vector according to claim 28 wherein the GABA_(B)-R1 receptor is aGABA_(B)-R1c receptor or variant thereof.
 32. A stable cell linecomprising a vector according to claim
 28. 33. The stable cell lineaccording to claim 32 which is a modified HEK293T cell line.
 34. Astable cell line modified to express both GABA_(B)-R1 and GABA_(B)-R2receptor proteins or variants thereof.
 35. The stable cell lineaccording to claim 34 which is a modified HEK293T cell line.
 36. AGABA_(B) receptor produced by the stable cell line of claim
 34. 37. Anantibody specific for the receptor as claimed in claim
 24. 38. A methodfor identification of a compound which exhibits GABA_(B) receptormodulating activity, comprising contacting the GABA_(B) receptor ofclaim 24 with a test compound and detecting modulating activity orinactivity.
 39. A compound which modulates GABA_(B) receptor activity,identifiable by a method according to claim
 38. 40. A method oftreatment or prophylaxis of a disorder which is responsive to modulationof GABA_(B) receptor activity in a mammal, which comprises administeringto said mammal an effective amount of a compound identifiable by themethod according to claim
 38. 41. The method according to claim 40wherein the disorder is a CNS disorder, a GI disorder, a lung disorderor a bladder disorder.
 42. The method according to claim 40 wherein thedisorder is spasticity, epilepsy, Alzheimer's disease, pain or anaffective or feeding disorder.
 43. Use of a compound identifiable by themethod according to claim 38 in a method of formulating a medicament fortreatment or prophylaxis of a disorder which is responsive to modulationof GABA_(B) receptor activity in a mammal.
 44. The use according toclaim 43 wherein the disorder is a CNS disorder, a GI disorder, a lungdisorder or a bladder disorder.
 45. The use according to claim 43wherein the disorder is spasticity, Alzheimer's disease, pain or anaffective or feeding disorder.
 46. A method of producing a GABA_(B)receptor comprising introducing into an appropriate cell line a suitablevector or vectors comprising nucleotide sequences encoding forGABA_(B)-R1 and GABA_(B)-R2 receptors or variants thereof, underconditions suitable for obtaining expression of the receptors orvariants.